Positive Expression Of Markers Research Articles

CyCadas: accelerating interactive annotation and analysis of clustered cytometry data.

Single cell profiling by cytometry has emerged as a key technology in biology, immunology and clinical-translational medicine. The correct annotation, which refers to the identification of clusters as specific cell populations based on their marker expression, of clustered high-dimensional cytometry data, is a critical step of the analysis. Its accuracy determines the correct interpretation of the biological data. Despite the progress in various clustering algorithms, the annotation of clustered data still remains a manual, time consuming and error-prone task. We developed a user-friendly cluster annotation and differential abundance detection tool that can be applied on data generated with Self Organizing Map clustering algorithms, thus simplifying the annotation process of datasets that consist of hundreds or thousands of clusters. We present Cytometry Cluster Annotation and Differential Abundance Suite (CyCadas), a semi-automated software tool that facilitates cluster annotation in cytometry data by offering both visual and computational guidance. CyCadas addresses the critical need for efficient and accurate annotation of high-resolution clustered cytometry data, significantly reducing the time needed to perform the analysis compared to both manual gating approaches and manual annotation of clustered data. The tool features a user-friendly interface, visual tools enabling data exploration and automated threshold estimation to separate negative and positive marker expression. It facilitates the definition and annotation of cell phenotypes among multiple clusters in a tree-based data structure. Finally, it calculates the abundance of various cell populations across the conditions with statistical interpretation. It is an ideal resource for researchers aiming to streamline their cytometry workflow. CyCadas is available as open source at: https://github.com/DII-LIH-Luxembourg/cycadas.

Efficient induction of pluripotent stem cells differentiated into mesenchymal stem cell lineages.

Mesenchymal stem cells (MSCs) have garnered significant attention in the field of cell-based therapy owing to their remarkable capabilities for differentiation and self-renewal. However, primary tissue-derived MSCs are plagued by various limitations, including constrained tissue sources, arduous and invasive retrieval procedures, heterogeneous cell populations, diminished purity, cellular senescence, and a decline in self-renewal and proliferative capacities after extended expansion. Addressing these challenges, our study focuses on establishing a robust differentiation platform to generate mesenchymal stem cells derived from induced pluripotent stem cells (iMSCs). To achieve this, we used a comprehensive methodology involving the differentiation of induced pluripotent stem cells into MSCss. The process was meticulously designed to ensure the expression of key MSC positive markers (CD73, CD90, and CD105) at elevated levels, coupled with the minimal expression of negative markers (CD34, CD45, CD11b, CD19, and HLA-DR). Moreover, the stability of these characteristics was evaluated across 10th generations. Our findings attest to the success of this endeavor. iMSCs exhibited robust expression of positive markers and limited expression of negative markers, confirming their MSC identity. Importantly, these characteristics remained stable even up to the 10th generation, signifying the potential for sustained use in therapeutic applications. Furthermore, our study demonstrated the successful differentiation of iMSCs into osteocytes, chondrocytes, and adipocytes, showcasing their multilineage potential. In conclusion, the establishment of induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) presents a significant advancement in overcoming the limitations associated with primary tissue-derived MSCs. The remarkable stability and multilineage differentiation potential exhibited by iMSCs offer a strong foundation for their application in regenerative medicine and tissue engineering. This breakthrough paves the way for further research and development in harnessing the full therapeutic potential of iMSCs.

594-P: Human Nrf2-Active Multipotent Stromal Cell Exosomes Promote Tissue Repair in a Wound Model of Type 2 Diabetes

Exosomes, nanosized extracellular vesicles, may be key to translating multipotent stromal cell (MSC) therapy to the bedside. We previously found that nuclear factor erythroid 2-related factor 2 (Nrf2) regulates MSC promotion of tissue repair in diabetes. Here, we explore a novel role of Nrf2 in exosome biogenesis and investigate whether exosome treatment recapitulates the effects MSCs have on wound healing in mice with type 2 diabetes. Exosomes were harvested by differential ultracentrifugation of conditioned human bone marrow derived MSC media. For Nrf2-active exosomes, MSCs were incubated with potent Nrf2 activator, CDDO-Im. MSC characterization demonstrated robust tri-lineage differentiation, >95% expression of positive markers (CD44/CD73/CD90/CD105/CD106), and <5% expression of negative markers (CD45/CD31/CD14/CD19/HLA-DR). Immunoblotting of exosome isolates demonstrates enrichment for CD81, CD9 and TSG101. Nanoparticle tracking analysis demonstrates that Nrf2-active MSCs increase exosome secretion by 54%, compared to Nrf2-baseline MSCs (p<0.05). Exosomes were injected into the wound margin of full-thickness wounds on Leprdb/db (db/db) mice. Both Nrf2-baseline and Nrf2-active exosome treatment significantly reduced closure time to 15.5 and 14 days respectively, compared to 29.8 days for vehicle-treated wounds (p<0.05). This reduction eliminated the delay in closure time compared to wounds of C57/B6 mice. Nrf2-active exosome treatment of db/db wounds reduced closure time by a further 2.6 days compared to untreated C57/B6 wounds. At day 10, exosome treated db/db wounds have significant decreases in epithelial gap, expanded granulation tissue, and greater density of CD31+ vessels compared to vehicle-treated wounds. We conclude that activating Nrf2 in MSCs increases isolation efficiency by amplifying exosome yield and content. MSC exosome based therapies have potential for rapid translation to improve clinical wound healing outcomes. Disclosure J. Kuhn: None. A. Hassan: None. S. Sharma: None. J. Kwong: None. M. Rahman: None. S. Adam: None. J. Lee: None. A.P. Villarreal Ponce: None. P.S. Rabbani: None. Funding Plastic Surgery Foundation; Wound Healing Society; New York University Clinical and Translational Science Institute

Immunohistochemical Analysis of Cancer Stem Cell Marker Expression in Papillary Thyroid Cancer.

Cancer stem cell (CSC) markers have prognostic significance in various cancers, but their clinical significance in papillary thyroid carcinoma (PTC) has not been demonstrated. In this study, CSC markers expressed in PTC and their relationships with prognosis were evaluated. We constructed tissue microarrays for 386 PTC cases, divided it into 42 low risk cases and 344 intermediate risk cases according to the American Thyroid Association 2009 Risk Stratification System. Immunohistochemical staining of CSC markers (CD15, CD24, CD44, CD166, and ALDH1A1) was performed, and the proportion of stained cells and immunostaining intensity were evaluated to determine positive marker expression. The relationships between CSC marker expression and other clinicopathological parameters or survival were analyzed. CD15 expression was higher in PTC with intermediate risk than in PTC with low risk (29.4 vs. 11.9%, p = 0.017). According to a multivariate analysis, CD15, CD44, CD166, and ALDH1A1 positivity were independently associated with a shorter progression-free survival (PFS) (odds ratio [OR]: 1.929, 2.960, 7.485, and 3.736; p = 0.016, p = 0.026, p < 0.001, and p = 0.006, respectively). Higher N and cancer stage were the only other clinical factors associated with a shorter PFS (OR: 2.953 and 1.898, p = 0.011 and p = 0.034). Overexpression of CSC markers in PTC was associated with shorter PFS during follow-up. Immunohistochemical staining of CSC markers may provide useful information for predicting patient outcomes.

Frequency and Clinical Characteristics Associated with Putative CAR Targets ADGRE2, CCR1, CD70, and LILRB2 in Acute Myeloid Leukemia

Background: ADGRE2, CCR1, CD70, and LILRB2 expressed on the surface of myeloid blasts but not normal hematopoietic stem cells, T cells or other tissues have been recently suggested as candidate chimeric antigen receptor (CAR) targets for engineered T cells in acute myeloid leukemia (AML) patients.Aim: To validate the expression pattern of the recently identified candidate CAR targets ADGRE2, CCR1, CD70, and LILRB2 on leukemic blasts in a cohort of newly diagnosed AML patients.Methods: 109 patients with de novo (n=87) or secondary (n=22) AML were included in the analysis. Patients were classified according to the 2008 WHO classification and cytogenetically characterized by chromosome banding analysis. Molecular analyses were performed by Sanger and next-generation sequencing (NGS) with a panel of 46 genes. Bone marrow or peripheral blood samples from the time of first diagnosis were obtained to perform multi-color flow cytometry analysis evaluating the expression levels of ADGRE2 (also known as EMR2 or CD132), CCR1 (also known as CD191), CD70 and LILRB2 (also known as CD85d) antigens on the surface of myeloid blasts. Patients with expression of the marker on ≥20% of blast cells were defined positive. Informed consent was obtained from all patients in accordance to the declaration of Helsinki and institutional guidelines.Results: ADGRE2, CCR1, CD70 and LILRB2 were expressed in 100%, 70%, 27.5%, 27.5% of patients with a median expression on myeloid blasts of 87.8% (range 30.4-99.8%), 43.9% (range 21.1-86.2%), 29.0% (range 20.0-55.4%), and 35.6% (range 20.7-90.6%), respectively. A subset analysis was performed to determine expression levels of the candidate targets in CD3 positive cells. Of patients with positive marker expression on blast cells ADGRE2, CCR1, CD70 and LILRB2 were expressed on 14.5% (range 0.0-64.2%), 19.9% (0.0-73.0%), 15.3% (range 0.0-35.1%), and 2% (range 0.1-18.4%) of CD3+ T cells, respectively. The proportion of patients with ≥80% expression on blasts was 61.5% (n=67), 0.9% (n=1) and 3.7% (n=4) for ADGRE2, CCR1 and LILRB2, respectively. Of those 53.7%, 0%, and 100% had marker expression <20% on CD3+ T cells and 15%, 0%, and 25% had no expression (<1%) of ADGRE2, CCR1 and LILRB2 on CD3+ T cells.There were no significant differences in the distribution of age, type of AML, sex, WHO subtypes, cytogenetic risk according to 2017 ELN classification, WBC count, hemoglobin and platelet count or for the type of consolidation treatment between CCR1, CD70, and LILRB2 positive compared to negative patients except for a higher platelet count in LILRB2 positive patients (p=0.009). To evaluate the association between mean expression level and mutational profile molecular analyses were performed as mentioned above and correlated with expression levels. More blast cells expressed CCR1 in CEBPA mutated patients compared to CEBPA wildtype patients (43.7% vs. 32.8%, p=0.003), while fewer blast cells expressed LILRB2 in IDH2 mutated compared to wildtype patients (9% vs. 16.8%, p=0.027). However, no other mutations correlated with the expression of the candidate CAR targets. Next, Pearson correlation was calculated to determine co-expression of the candidate CAR targets. A positive correlation was found between CCR1 and CD70 expression on blast cells (R=0.673, p<0.001). ADGRE2 correlated with LILRB2 and CD70 expression on CD3+ T cells (R=0.631, p<0.001 and R=0.416, p<0.001, respectively). CCR1 correlated with CD70 and LILRB2 on CD3+ T cells (R=0.807, p<0.001 and R=0.46, p<0.001, respectively). The rate of complete remission was similar for patients with and without expression of CCR1, CD70 and LILRB2 (82.2% vs. 14.5%, p=0.115; 73.3% vs. 20%, p=0.349; 83.3% vs. 13.3%, p=0.828, respectively). Overall survival, event-free and relapse-free survival were also similar for patients with and without expression of CCR1, CD70 and LILRB2.Conclusions: Our data show variable expression levels of candidate CAR targets in AML blast cells with ADGRE2 being expressed at high levels in all patients. However, expression levels were not specifically associated with patient characteristics or outcome. Our findings favor ADGRE2 as potentially suitable for CAR targeting as it had the most favorable expression profile on blasts and T cells in AML patients. DisclosuresKoenecke:BMS: Consultancy; Amgen: Consultancy; abbvie: Consultancy; Roche: Consultancy. Fiedler:Daiichi Sankyo: Other: support for meeting attendance; Gilead: Other: support for meeting attendance; Amgen: Other: support for meetíng attendance; Pfizer: Research Funding; Amgen: Research Funding; Amgen: Patents & Royalties; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; ARIAD/Incyte: Membership on an entity's Board of Directors or advisory committees, support for meeting attendance; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSO: Other: support for meeting attendance; Teva: Other: support for meeting attendance; JAZZ Pharmaceuticals: Other: support for meeting attendance. Ganser:Novartis: Membership on an entity's Board of Directors or advisory committees.

187 Morphological Appearance and Expression of Spermatogonial Stem Cell Markers in White Rhinoceros Testicular Tissue

Spermatogonial stem cells (SSC) have been isolated from testicular tissue (TT) of several mammalian species and differentiated into mature spermatozoa following transplantation or in vitro culture. The northern white rhinoceros (NWR; Ceratotherium simum cottoni) is critically endangered. Thus, frozen NWR TT, cryopreserved and stored at the San Diego Zoo’s Frozen Zoo®, potentially contain SSC that could be a source of spermatozoa. The method used for cryopreserving TT may affect the integrity and number of SSC. Therefore, identifying alterations in the seminiferous tubules (ST) of frozen-ndash;thawed-NWR TT will provide insight into the condition of the SSC. Therefore, our aims were to (1) determine the effect of freezing rhinoceros TT on the structure of epithelium, and (2) identify SSC (GFRα1, GPR125) and pluripotent (SSEA-4 and Oct-4) markers. Testicular tissue of an adult NWR and a stillborn southern white rhinoceros (SWR) were frozen by equilibration of TT for 30 min at 4.0°C in PBS and 1.5 M dimethyl sulfoxide (DMSO), cooled at 2.0°C/min to −4.0°C, 0.3°C/min to −40°C, and plunged into liquid nitrogen. Tissues were thawed at 37°C in a water bath and DMSO removed in a 4-step dilution. Tissue was then fixed, dehydrated, and paraffin embedded. For morphological evaluations, frozen-ndash;thawed tissue was sectioned and stained with hematoxylin and eosin (H&amp;E). The TT from both rhinoceros collected immediately after death (fresh) and stained with H&amp;E were used as a control for cryopreservation. Localization of SSC and pluripotent markers in ST of frozen-ndash;thawed TT was detected by immunohistochemistry. Morphologically, fresh-NWR TT was severely altered, displaying large epithelium gaps and partial (62.2%) or total detachment (37.7%) from, and slight damage (35.5%) to, the basement membrane. The number of pyknotic nuclei per ST was moderate (15.6 ± 7.2%). Many of these changes could have resulted from autolysis and handling before tissue preparation. In contrast, histological appearance of fresh-SWR was good, with 98.3% of the tubules intact, and a small proportion of pyknotic cells (0.8 ± 1.5%). Seminferous tubule (n = 30/male) length and width (μm; ± SEM) differed between NWR (635.2 ± 34.4 × 214.6 ± 10.8) and SWR (277.7 ± 13.8 × 73.2 ± 2.4; P &lt; 0.05). Damages after cryopreservation compared with fresh tissue comprised (1) epithelium detachment, NWR = 100% (P &lt; 0.0001), and SWR = 43.3% (P &lt; 0.001); (2) basement membrane alteration, only in NWR (93.0%; P &lt; 0.001); and (3) decreased length and width in the ST, NWR = 409.4 ± 18.1 × 173.4 ± 8.2 (P &lt; 0.05), and SWR = 195.2 ± 8.3 × 61.6 ± 2.8 (P &lt; 0.05), with loss of lumen in both males. Immunohistochemistry revealed that NWR expressed GFRα1 and GPR125 at various stages of spermatogenqaesis, whereas Oct-4 was detected in few cells. In contrast to NWR, Oct-4 expression in SWR was located at the basement membrane; SSEA-4 was not detected in either male. In conclusion, freezing-induced morphological alterations in rhinoceros ST and positive expression of markers for SSC and pluripotency suggest the presence of SSC. Further studies are required to evaluate the viability of rhinoceros SSC.

Manufacturing of Mesenchymal Stromal Cells for the Treatment of Graft-Versus-Host Disease: A Survey within the European Society of Blood and Marrow Transplantation

The immunosuppressive properties of mesenchymal stromal cells (MSC) have been widely exploited in the clinical setting to control severe graft-versus host disease (GvHD). Although not randomized, several studies have convincingly shown that MSC can induce good clinical responses, and patients who respond exhibit much improved survival. However, the factors predictive of clinical responses have not yet been identified, and what may complicate the interpretation of the results is the potential heterogeneity of the MSC preparation, especially in the absence of any validated potency assay. Clinical scale manufacturing of therapeutic MSC is performed according to different protocols that vary for MSC source (bone marrow, adipose tissue, umbilical cord blood or tissue), extraction methods, expansion protocols and product specificity.A harmonized protocol for the production and product specification would be therefore beneficial to better compare the results obtained amongst different Centers.In order to acquire information on the variability in the MSC manufacturing process we have sent a questionnaire tothe European Society for Blood and Marrow Transplantation (EBMT) Centers registered as producing MSC. Initial data from 15 centers were obtained and analyzed. The majority of centers manufacture MSC from bone marrow (88%), whilst only 2 centers produce MSC from umbilical cord blood or cord tissue. One of the major changes in the manufacturing process has been the replacement of fetal bovine serum with human platelet lysate as a medium supplement. 90% of centers currently use platelet lysate, either commercially available or from pools of expired platelets obtained from the blood bank (Figure 1).60% of the centers use only allogeneic MSC, whilst one center manufactures only autologous MSC,and 67% of these facilities administer MSC exclusively from frozen batches. Aside from variations in the culture method, there is a large heterogeneity also regarding product specification. Phenotypical characterization represents a fundamental release criterion for all centers, and all surveyed facilities conduct analysis for the expression of CD73, CD90 and CD105, and CD45 through flow cytometry. However, the threshold of marker expression that is accepted as a release criterion is highly variable, as the acceptance value for CD45 expression can be set at less than 20%, 10%, 5%, 2% or 1% (Figure 2). Similarly, threshold percentage of positive marker expression varies from 70% to a very stringent 95%. Furthermore, the addition of several other markers to the negative and positive panel of expression for product specification is again unsystematic, with a high variability in the release threshold levels and in the type of markers analyzed. Most centers add CD19, CD34, CD14, HLA-DR and CD3 negativity to the panel, and only 2 centers check expression of CD166. Alongside product specification meeting sterility and mycoplasma free product, 73% of centers checks for presence of endotoxin in the final formulation. The majority of centers (67%) perform karyotyping, whilst only 27% analyze the immunosuppressive activity of MSC against mitogen activated T cells, and only 33% of the centers determine differentiation ability of MSC into adipocyte, osteoblasts and chondrocytes. These tests are not always performed in parallel, as only 2 centers test tri-lineage differentiation and karyotyping, 3 centers analyze anti-proliferation activity in conjunction with karyotype and 1 center examines differentiation alongside immunosuppressive activity.The initial data collected from this survey indicate that there is a high variability of culture method, supplement material and, even more importantly, difference in acceptance criteria for release of MSC as clinical product. Furthermore, many of these criteria should be extensively revised as they are not entirely substantiated by scientific evidence. This survey should be considered the first step towards harmonization, which should bring to a better interpretation of clinical responses in patients in combination with scientific findings coming from pre-clinical models and in vitro studies. [Display omitted] [Display omitted] [Display omitted] DisclosuresApperley:Bristol Myers Squibb: Honoraria, Speakers Bureau; Incyte: Speakers Bureau; Ariad: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau. Kuball:Gadeta B.V,: Membership on an entity's Board of Directors or advisory committees. Bonini:Molmed SpA: Consultancy; TxCell: Membership on an entity's Board of Directors or advisory committees.

Centrifugal gravity-induced BMP4 induces chondrogenic differentiation of adipose-derived stem cells via SOX9 upregulation.

BackgroundCartilage does not have the capability to regenerate itself. Therefore, stem cell transplantation is a promising therapeutic approach for impaired cartilage. For stem cell transplantation, in vitro enrichment is required; however, stem cells not only become senescent but also lose their differentiation potency during this process. In addition, cytokines are normally used for chondrogenic differentiation induction of stem cells, which is highly expensive and needs an additional step to culture. In this study, we introduced a novel method to induce chondrogenic differentiation of adipose-derived stem cells (ASCs), which are more readily available than bone marrow-derived mesenchymal stem cells(bMSCs), using centrifugal gravity (CG).MethodsASCs were stimulated by loading different degrees of CG (0, 300, 600, 1200, 2400, and 3600 g) to induce chondrogenic differentiation. The expression of chondrogenic differentiation-related genes was examined by RT-PCR, real-time PCR, and western blot analyses. The chondrogenic differentiation of ASCs stimulated with CG was evaluated by comparing the expression of positive markers [aggrecan (ACAN) and collagen type II alpha 1 (COL2A1)] and negative markers (COL1 and COL10) with that in ASCs stimulated with transforming growth factor (TGF)-β1 using micromass culture, immunofluorescence, and staining (Alcian Blue and Safranin O).ResultsExpression of SOX9 and SOX5 was upregulated by CG (2400 g for 30 min). Increased expression of ACAN and COL2A1 (positive markers) was detected in monolayer-cultured ASCs after CG stimulation, whereas that of COL10 (a negative marker) was not. Expression of bone morphogenetic protein (BMP) 4, an upstream stimulator of SOX9, was upregulated by CG, which was inhibited by Dorsomorphin (an inhibitor of BMP4). Increased expression of proteoglycan, a major component of cartilage, was confirmed in the micromass culture of ASCs stimulated with CG by Alcian Blue and Safranin O staining.ConclusionsChondrogenic differentiation of ASCs can be induced by optimized CG (2400 g for 30 min). Expression of SOX9 is upregulated by CG via increased expression of BMP4. CG has a similar ability to induce SOX9 expression as TGF-β1.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0445-6) contains supplementary material, which is available to authorized users.

534 Vitiligo: Studying the dermal compartment

Homeostasis of pigmentary system is regulated by strictly balanced interactions at both molecular and cellular level and a multitude of messengers and growth factors released by epidermal and dermal cells regulate melanocyte activities and consequently skin pigmentation. Vitiligo is characterized by the progressive disappearance of melanocytes. Increasing evidences are currently highlighting an intrinsic metabolic impairment of melanocytes, favouring the increase of intracellular oxidative stress, which, in turn, may promote the onset of a premature senescent-like phenotype. Despite the important role of the dermis in regulating melanocyte physiology, few data have evaluated its involvement in vitiligo. The aim of our study was to investigate the dermal compartment of non lesional vitiligo skin, focusing on the features of extracellular matrix proteins and fibroblasts. We employed primary cultures of non lesional vitiligo fibroblasts and performed a parallel ex vivo evaluation on skin biopsies collected from the same patients from whom the cells were derived. Our results demonstrate that vitiligo fibroblasts display increased basal ROS levels associated with the up-modulation of stress-induced markers, e.g. p53 and its target gene GADD45. The higher intrinsic oxidative stress favours the transition of fibroblasts into a myofibroblasts-like phenotype, as assessed by the positive expression of markers related to myofibroblasts including alpha-smooth muscle actin, the glycoprotein fibronectin, the intermediate filament associated protein vimentin and the secretory proteins interleukin IL-6 and hepatocyte growth factor. The results point to the involvement of the entire skin in vitiligo, showing the presence of modifications not restricted to melanocytes, which constitute the long-established major player of the disease, but extended to the dermis. The focus on normal appearing skin may therefore allow to recognize the appearance of cellular phenomena before the clear clinical onset of the disease and possibly restrict the spread of the lesions.

Multiparameter flow cytometry for the characterization of human embryonic stem cells

Using multiparameter staining methods and flow cytometry to investigate the pluripotency of HUES7 human embryonic stem cell cultures, it was found that the multidimensional approach of marker co-expression allowed the different cell populations to be easily identified and demonstrated cross reactivity between the SSEA 4 and SSEA 1 antibodies, resulting in a substantial false positive SSEA 1 population. It is the accepted norm to apply control gates at a 95% confidence level of the isotype control; however, this study found that adjusting the control gate to a 99% confidence level significantly reduced the effect of this cross reactivity. Though conversely, this gating shift also decreased the positive marker expression of SSEA 4 and Tra-1-60, indicating that there is a need for strongly expressing markers coupled with increased optimization of fluorophore/antibody combinations before a gating strategy of 99% can be implemented on a more routine basis.

212 PORCINE BONE MARROW-DERIVED MESENCHYMAL STEM CELLS DIFFERENTIATE IN VITRO INTO SMOOTH MUSCLE CELLS.

In the context of multipotent stem cells, mesenchymal stem cells (MSC) derived from bone marrow have been identified as most promising cell types for the treatment of smooth muscle related injured tissues and organs. In the present study, the ability of porcine bone marrow derived MSC to differentiate in vitro into smooth muscle cells (SMC) was examined. MSC were isolated from domestic pig bone marrow by their readily adherent property to tissue culture plastic with fibroblast-like morphology. Cells were analysed for the expression of MSC specific markers by flow cytometer and mesenchymal lineage differentiation by following previously published protocols. Differences in values were analysed by one-way ANOVA using SPSS and data are presented as mean ± SD. Flow cytometry analysis of MSC showed the positive expression of markers, such as CD29 (97.33 ± 2.08%), CD44 (97.67 ± 1.15%), CD73 (62.33 ± 2.89%), CD90 (96.67 ± 2.08%) and vimentin (59.33 ± 2.52%). In contrast, the expression levels were significantly lower for CD34 (3.33 ± 1.53%), CD45 (3.67 ± 1.53%), major histocompatibility complex class II (MHC class II, 10.33 ± 2.52%) and swine leukocyte antigen-DR (SLA-DR, 9.67 ± 2.08%). The MSC were further confirmed by their ability to differentiate in vitro along the distinct lineages of adipocytes (Oil red O), osteocytes (von Kossa and Alizarin red) and chondrocytes (Alcian blue). Induction of SMC differentiation was performed with supplementation of porcine transforming growth factor-β (TGF-β) and recombinant human bone morphogenic protein 4 (BMP4) as described earlier (Wang et al. 2010 Tissue Eng. A 1201–1213) with minor modifications. Upon induction, porcine MSC acquired myoblast-like morphology with intracellular thin filaments. Immunofluorescence staining showed the presence of early and late markers of smooth muscle differentiation, such as α-smooth muscle actin (α-SMA), calponin, smooth muscle 22 α (SM22α) and smooth muscle-myosin heavy chain (SM-MHC) and their expression levels varied from 22.65% to 56.75%. Later, the expression of selected markers was demonstrated by Western blotting analysis. Consistent with this phenotypic characterisation, reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR (RT-qPCR) further showed the expression and a sequential up-regulation of transcripts for α-SMA, calponin, SM22α and SM-MHC. However, no expression of SMC-specific markers was observed in untreated MSC. In conclusion, these findings suggest the ability of porcine MSC from bone marrow to differentiate in vitro into SMC in the presence of growth factors. Further understanding of SMC differentiation with functional properties would be essential for employing porcine MSC as a useful model for cell-based tissue engineering and regeneration strategies. This work was supported by Basic Science Research Program through the National Research Foundation (NRF) funded by the Ministry of Education, Science and Technology (2010-0010528) and BioGreen 21 (20070301034040), Republic of Korea.

Generation of human induced pluripotent stem cells from osteoarthritis patient-derived synovial cells

This study was undertaken to generate and characterize human induced pluripotent stem cells (PSCs) from patients with osteoarthritis (OA) and to examine whether these cells can be developed into disease-relevant cell types for use in disease modeling and drug discovery. Human synovial cells isolated from two 71-year-old women with advanced OA were characterized and reprogrammed into induced PSCs by ectopic expression of 4 transcription factors (Oct-4, SOX2, Klf4, and c-Myc). The pluripotency status of each induced PSC line was validated by comparison with human embryonic stem cells (ESCs). We found that OA patient-derived human synovial cells had human mesenchymal stem cell (MSC)-like characteristics, as indicated by the expression of specific markers, including CD14-, CD19-, CD34-, CD45-, CD44+, CD51+, CD90+, CD105+, and CD147+. Microarray analysis of human MSCs and human synovial cells further determined their unique and overlapping gene expression patterns. The pluripotency of established human induced PSCs was confirmed by their human ESC-like morphology, expression of pluripotency markers, gene expression profiles, epigenetic status, normal karyotype, and in vitro and in vivo differentiation potential. The potential of human induced PSCs to differentiate into distinct mesenchymal cell lineages, such as osteoblasts, adipocytes, and chondrocytes, was further confirmed by positive expression of markers for respective cell types and positive staining with alizarin red S (osteoblasts), oil red O (adipocytes), or Alcian blue (chondrocytes). Functional chondrocyte differentiation of induced PSCs in pellet culture and 3-dimensional polycaprolactone scaffold culture was assessed by chondrocyte self-assembly and histology. Our findings indicate that patient-derived synovial cells are an attractive source of MSCs as well as induced PSCs and have the potential to advance cartilage tissue engineering and cell-based models of cartilage defects.

Abstract C25: Enabling in situ quantitative biomarker analysis in the context of tissue morphology

Abstract Modern personalized medicine makes use of individual molecular biomarkers to subdivide tumors into subsets that behave differently from each other. A large number of clinical trials are evaluating several novel therapeutic targets and relevant biomarkers. Tissue biomarkers are gaining prominent position in drug discovery and development programs and are commonly included in clinical trial testing to identify patients that would profit from particular treatments, measure drug efficacy and/or monitor the success of therapeutic interventions. The demands on accuracy and reliability of tests are very high. The quality of tissue biomarker testing can be affected by various pre-analytic factors (time to fixation, time of fixation, type of fixation, method of tissue processing), analytic factors (assay validation, equipment validation, test reagents, standardized control materials) and post-analytic factors (interpretation criteria, use of image analysis, quality assurance procedures). In this poster we will focus on analytic and post-analytic aspects, and more specifically - imaging cytometry as a method of choice in automated analysis of tissue and tissue microarray specimens. Materials and Methods: 1. Standard cytometric QC materials - for within-run precision of fluorescence measurements, Flow Check beads (Beckman-Coulter), for run-to-run precision of fluorescence measurements, CaliBrite Beads (B.D. Biosciences), for run-to-run precision of chromatic staining, negative and positive punch cores of breast tissue labeled for HER2/Neu with CB11 antibody developed with DAB and counterstained with Hematoxylin. 2. Tissue microarrays - whole mouse, rat and major human organ TMAs (Invitrogen).Arrays stained with dual IHC protocols for CD31/CD34, cytokeratin 7/CD31, S100/GFAP, kappa/lambda (in situ hybridization) and Ki67/CD68. 3. Specimens were analyzed on an iCys® Imaging Cytometer (CompuCyte Corporation, Westwood, MA). Quality control measures and Results: a) within-run precision of fluorescence measurement - a Coefficient of Variation (CV) of fluorescence intensity of less than 3% measured over a large bead population; b) run-torun precision of fluorescence measurements, the mean of fluorescence intensity measurements within +/− 2 Standard Deviations (SDs) as established by baseline runs; c) run-to-run precision of chromatic staining - the mean DAB laser light loss measurements within +/− 2 Standard Deviations (SDs) as established by baseline runs. TMA analysis: instrument automatically identified core elements, performed spectral deconvolution to isolate markers of interest. Chromatic measurements by laser-light-loss and fluorescence measurements were obtained simultaneously and combined to quantify the expression of the markers of interest. High resolution images ofthe cores were obtained simultaneously with the quantitative analytical data. Quantitative end-points on a per-core basis included: percent positive marker expression, mean expression level, total marker expression level and co-localization of markers. The technique of “image masking” effectively reduced the background levels in tissue elements. Summary: LSC technology provides accurate and reproducible measurements of tissue biomarkers in unbiased, highly quantitative manner. It is well suited for tissue-based biomarker discovery and clinical trials. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C25.

Flow immunocytochemistry of marker expression in cells from body cavity fluids

Diagnostic cytology based on the examination of cells from body cavity fluids misses approximately 50% of patients with a proven malignancy. In an earlier study, we used immunohistochemical detection of epithelial membrane antigen expression with flow cytometric detection of DNA aneuploidy to reduce the number of false negatives. In the present study, we have combined DNA flow cytometry with flow cytometric detection of marker expression to analyze cells from body cavity fluids. Seventy-nine specimens of ascites and pleural fluids were analyzed by diagnostic cytology, DNA flow cytometry, and for the expression of the following markers: Ber-EP4, progesterone (PR), MUC4, and thyroid transcription factor-1 (TTF-1). DNA index of equal to or greater than 1.2 was seen in 33/79 (41.7%) of the samples. Statistical analysis of 79 samples in which data from cytology, DNA aneuploidy, and expression of at least one of the markers was available showed that by combining data from positive marker expression with that of aneuploidy, the sensitivity was increased from 58.5 to 100%. In contrast, out of the 38 samples designated as non-malignant by diagnostic cytology, nine had aneuploid DNA content and 16 of the diploid samples had a positive marker expression. Specificity was reduced from 74.7 to 31.6% due to the presence of aneuploidy and marker expression in these samples. ALDH1(pos)/CD44(pos)/CD24(neg) expression has been reported to be associated with human breast tumor stem cells. Some of our samples had cells with this phenotype. Flow cytometry offers the advantage of rapid multiparametric analysis of DNA aneuploidy and marker expression in cells from body cavity fluids based on the analysis of a large number of cells without observer bias. By further developing the use of specific markers and aneuploidy, it may be possible to refine flow cytometric analysis for rapid detection of malignant cells in body cavity fluids.

Soy Consumption and Histopathologic Markers in Breast Tissue Using Tissue Microarrays

This study examined the relation of soy intake with hormonal and proliferation markers in benign and malignant breast tissue using tissue microarrays (TMAs). TMAs with up to 4 malignant and 4 benign tissue samples for 268 breast cancer cases were constructed. Soy intake in early life and in adulthood was assessed by questionnaire. The TMAs were stained for estrogen receptor (ER) α, ERβ, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2/neu), proliferating cell nuclear antigen (PCNA), and Ki-67 using standard immunohistochemical methods. Logistic regression was applied for statistical analysis. A higher percentage of women showed positive marker expression in malignant than in benign tissue. With one exception, HER2/neu, no significant associations between soy intake and pathologic markers were observed. Early life soy intake was associated with lower HER2/neu and PCNA staining of malignant tissue. In benign tissue, early life soy intake showed higher ER and PR expression, but no difference in proliferation markers. The results of this investigation provide some assurance that soy intake does not adversely affect markers of proliferation. TMAs were shown to be a useful tool for epidemiologic research.

Differential Expression of Ki-67 and Sex Steroid Hormone Receptors Between Genders in Peritoneal Mesothelioma

Gender influence on survival in mesothelioma has been observed in several large clinical series. However, this gender effect has not been investigated. Female patients often have less aggressive tumors and survive longer. However, few studies in the literature have explained the molecular basis of this finding. Understanding this difference at a molecular level may offer the hope of improving survival via hormonal manipulation.We investigate the expression of Ki-67 and sex steroid receptors; estrogen receptors (ER), progesterone receptors (PR) and androgen receptors (AR) to elucidate any pathognomonic difference that characterize this gender difference. Positive expression of markers was observed in 95% (Ki-67), 80% (ER), 100% (PR) and 65% (AR) of patients. Expression of markers between gender showed a higher Ki-67 in males (M = 1.3%, F = 0.6%), higher estrogen receptor in females (M = 0.6%, F = 1.7%) and higher progesterone receptor in females (M = 1.0%, F = 1.4%). Twenty patients were treated with cytoreductive surgery and hyperthermic intraperitoneal chemotherapy in our peritonectomy unit. Paraffin sections of the tumor specimens were retrieved for immunohistochemical analysis. The immunostaining was performed using monoclonal mouse anti-human antibodies on an autostainer (Autostainer Plus; Dako, Inc.). The intensity of the stains were quantified using the Image-Pro Plus (IPP) 4.5 (Media Cybernetics, Silver Spring, MD). For the first time, we demonstrate the presence of sex steroid receptors in peritoneal mesothelioma. Once the exact functional effects of these receptors are understood, the use of established therapeutic options that are clinically available to target the sex steroid pathway may become a reality.

Expression of cytokeratin19, galectin-3 and HBME-1 in thyroid lesions and their differential diagnoses

To study immunohistochemical expression of cytokeratin19 (CK19), galectin-3 (Gal-3) and HBME-1 in thyroid lesions and to assess their usefulness as markers in the differential diagnoses of thyroid nodular lesions. Immunohistochemical staining was performed on formalin-fixed paraffin-embedded tissue of 21 cases of nodular goiters, 14 cases of toxic goiters, 15 cases of follicular adenomas (FA), 13 cases of follicular carcinomas (FC), 13 cases of follicular variant papillary carcinomas (FVPC) and 48 cases of classic papillary carcinomas (CPC). All three markers were expressed in the cytoplasm with no or weak expression in benign lesions and diffuse and strong in malignant cases. Positive expressions of CK19, Gal-3 and HBME-1 were present in 11of 21, two of 21, four of 21 in nodular goiters, seven of 14, one of 14, one of 14 in toxic goiters, nine of 15, two of 15, two of 15 in FA, 10 of 13, eight of 13, seven of 13 in FC, 13 of 13, 11 of 13, 12 of 13 in FVPC, and 48 of 48, 45 of 48, 46 of 48 in CPC. The expression rates of the three markers between benign lesions (nodular goiters, toxic goiters and FA) and malignant lesions (FA, FVPC and CPC) were statistically significant. Among the three follicular lesions (FA, FC and FVPC), the differences were statistically significant as well. Nine, seven and six cases were negative for all three markers in nodular goiters, toxic goiters and FA, respectively. Only one case in FC was negative for all three markers, no case was all negative in FVPC and CPC; the rate of one case with two or more positive marker expression in nodular goiters, toxic goiters, FA, FC, FVPC and PC was 14.2% (3/21), 21.43% (3/14), 20.0% (3/15), 69.2% (9/13), 92.3% (12/13), 100.0% (48/48), the differences between benign lesions and malignant lesions and between FA, FC and FVPC were also statistically significant. Immunohistochemical stains of CK19, Gal-3 and HBME-1, especially when used in combination, can be an important adjunct to the histopathological diagnoses of thyroid lesions.