Early Passage Cells Research Articles

Human papillomavirus oncogenes reprogram the cervical cancer microenvironment independently of and synergistically with estrogen

High-risk human papillomaviruses (HPVs) infect epithelial cells and are causally associated with cervical cancer, but HPV infection is not sufficient for carcinogenesis. Previously, we reported that estrogen signaling in the stromal tumor microenvironment is associated with cervical cancer maintenance and progression. We have now determined how HPV oncogenes and estrogen treatment affect genome-wide host gene expression in laser-captured regions of the cervical epithelium and stroma of untreated or estrogen-treated nontransgenic and HPV-transgenic mice. HPV oncogene expression in the cervical epithelium elicited significant gene-expression changes in the proximal stromal compartment, and estrogen treatment uniquely affected gene expression in the cervical microenvironment of HPV-transgenic mice compared with nontransgenic mice. Several potential estrogen-induced paracrine-acting factors were identified in the expression profile of the cervical tumor microenvironment. The microenvironment of estrogen-treated HPV-transgenic mice was significantly enriched for chemokine/cytokine activity and inflammatory and immune functions associated with carcinogenesis. This inflammatory signature included several proangiogenic CXCR2 receptor ligands. A subset of the same CXCR2 ligands was likewise increased in cocultures of early-passage cells from human cervical samples, with levels highest in cocultures of cervical fibroblasts and cancer-derived epithelial cells. Our studies demonstrate that high-risk HPV oncogenes profoundly reprogram the tumor microenvironment independently of and synergistically with estrogen. These observations illuminate important means by which HPVs can cause cancer through alterations in the tumor microenvironment.

Abstract 3030: Mutant p53-driven metastasis explored utilizing a traceable, conditional osteosarcoma model

Abstract TP53 is the most frequently mutated gene in cancer and missense mutations in particular account for the majority of mutations. Mutant p53 proteins exert oncogenic Gain-of-Function (GOF) properties that contribute to increased migration and invasion in culture and metastasis in vivo. To elucidate how mutant p53 drives metastasis, we developed a traceable somatic osteosarcoma model that initiates with a single p53 mutation (p53R172H or p53R245W), or p53 loss specifically in osteoblasts using Osx-cre. To mark tumor cells, we utilized the mTmG allele which expresses membrane-targeted Tomato (mT) prior to Cre-mediated excision and membrane-targeted green fluorescent protein (mG) after excision so the tumor cells become GFP+ and the stromal cells remain RFP+. In this high penetrant, short latency mouse model, tumor micro-metastasis can be detected by GFP expression in the context of a normal stroma and immune system. The cohort has developed osteosarcoma with complete penetrance and an average survival of 304± 65 day. We observed metastasis in 55% of p53 mutant osteosarcomas which was significantly higher than 26% metastasis observed in p53 null tumors (p<0.01). In p53 mutant osteosarcomas, the metastasis rate was found significantly associated with low p53 copy number. Spectral karyotyping (SKY) on early passage cells derived from somatic p53 mutant tumors exhibited marked aneuploidy with modal chromosome numbers near tetraploid. To characterize the transcriptional profile associated with mutant p53 GOF, we analyzed and compared the RNA-Seq transcriptomes of eight tumors derived from p53R172H mutant mice with eight p53 null tumors. Comparative analysis of gene expression identified a signature of snRNA genes to be strongly associated with mutant p53 gain of function. Functional annotation of upregulated genes in mutant tumors was also highly enriched for chromatin modifying enzymes. These findings contribute to our understanding of the role of mutant p53 GOF in metastasis. Our long term goal is to study how tumor-stromal interactions affect tumor development and metastasis. This understanding will have broad translational significance in diagnosis and treatment of tumors with mutant p53. Citation Format: Rasoul Pourebrahimabadi, Yun Zhang, Mark J. McArthur, Guillermina Lozano. Mutant p53-driven metastasis explored utilizing a traceable, conditional osteosarcoma model [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 3030. doi:10.1158/1538-7445.AM2017-3030

Abstract 2206: Proteome phenotype of stage III metastatic melanoma and response to MEK inhibition

Abstract Introduction: Melanoma accounts for 5% of all skin cancers, yet is the most common cause of skin cancer-related deaths, and is the commonest lethal malignancy in young people (<40yrs). Patients with BRAF mutant melanoma show survival benefit from MAPK pathway inhibition, although response rates vary. We conducted proteomic screening using mass spectrometry to detect cellular processes that might predict response to MEK inhibition more effectively than genotyping alone. Methods: Protein extracts from early passages of ten AJCC Stage III local lymph node metastatic melanoma cell lines with known MAPK mutational status (BRAFmut, NRASmut, MAPKwt,) were used. Further, 32 fresh frozen NRASmut or BRAFmut AJCC Stage IIIC metastatic melanoma (lymph node metastases) specimens were obtained from patients with varying survival. Lysed cells and tissue were digested with trypsin and analysed by LC-MS/MS using a TripleTOF 5600/6600 mass spectrometer. Data-independent acquisition (or SWATH-MS) was used to match multiplexed MS/MS spectra against a spectral ion library enabling relative protein quantification. Cell line viability in the presence of 2µM AZD6244 (MEKi) was determined using a PrestoBlue assay. Results: The SWATH-MS approach enabled quantification of ~2500 proteins across all 10 cell lines. Principal component analysis clearly segregated melanomas based on in vitro sensitivity to MEK inhibition, whereas genotype alone did not. In total, we show 57 proteins whose abundances correlate (r²>0.75) with response to MEKi, revealing roles in cell pigmentation biosynthesis, lipid metabolism, adherence and inter-cell communication. Survival analysis demonstrated that patients whose cell lines were responsive to MEKi had lower mortality than patients whose cells were resistant to MEKi (survival >7.5 years MEKi sensitive versus <1.7 years MEKi resistant, p=0.01). Across the 32 tumour samples, ~2000 proteins were quantifiable. The proteome phenotype associated with MEK inhibition observed in patient cell lines was a dominant signal in fresh frozen stage III metastatic melanoma specimens, suggesting these patients may have profited from MAPK pathway inhibition had these drugs been available. In this patient cohort neither the genotype nor proteome phenotype correlated with survival. Conclusion: Proteomic profiling by mass spectrometry demonstrated a MEK inhibition phenotype in early passage cell lines and fresh frozen melanoma metastases which may provide utility for selecting patients to treat with MEK drugs. Citation Format: Christoph Krisp, Robert Parker, Dana Pascovici, James Wilmott, John F. Thompson, Graham J. Mann, Richard A. Scolyer, Nicholas K. Hayward, Mark P. Molloy. Proteome phenotype of stage III metastatic melanoma and response to MEK inhibition [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 2206. doi:10.1158/1538-7445.AM2017-2206

Efficacy of Anti-mesothelin Immunotoxin RG7787 plus Nab-Paclitaxel against Mesothelioma Patient-Derived Xenografts and Mesothelin as a Biomarker of Tumor Response.

Purpose: The purpose of this study was to evaluate the antitumor efficacy of the reduced immunogenicity anti-mesothelin immunotoxin RG7787 plus nab-paclitaxel against primary mesothelioma cell lines and tumor xenografts and the utility of mesothelin as a biomarker of tumor response.Experimental Design: Early-passage human malignant mesothelioma cell lines NCI-Meso16, NCI-Meso19, NCI-Meso21, and NCI-Meso29 were evaluated for sensitivity to RG7787 or nab-paclitaxel alone or in combination. In addition, the antitumor activity of RG7787 plus nab-paclitaxel was evaluated using NCI-Meso16, NCI-Meso21, and NCI-Meso29 tumor xenografts in immunodeficient mice. Serum mesothelin was measured at different time points to determine whether its levels correlated with tumor response.Results: All four primary mesothelioma cell lines highly expressed mesothelin with 41 × 103 to 346 × 103 mesothelin sites per cell and were sensitive to RG7787, with IC50 ranging from 0.3 to 10 ng/mL. Except for NCI-Meso19, these cells were also sensitive to nab-paclitaxel, with IC50 of 10 to 25 ng/mL. In vitro, RG7787 plus nab-paclitaxel led to decreased cell viability compared with either agent alone. In NCI-Meso16 tumor xenografts, treatment with RG7787 plus nab-paclitaxel led to sustained complete tumor regressions. Similar antitumor efficacy was observed against NCI-Meso21 and NCI-Meso29 tumor xenografts. In all three tumor xenograft models, changes in human serum mesothelin correlated with response to therapy and were undetectable in mice with complete tumor regression with RG7787 and nab-paclitaxel.Conclusions: RG7787 plus nab-paclitaxel is very active against primary human mesothelioma cells in vitro as well as in vivo, with serum mesothelin levels correlating with tumor response. These results indicate that this combination could be useful for treating patients with mesothelioma. Clin Cancer Res; 23(6); 1564-74. ©2016 AACR.

Genomic complexity and targeted genes in anaplastic thyroid cancer cell lines.

Anaplastic thyroid cancer (ATC) is a highly malignant disease with a very short median survival time. Few studies have addressed the underlying somatic mutations, and the genomic landscape of ATC thus remains largely unknown. In the present study, we have ascertained copy number aberrations, gene fusions, gene expression patterns, and mutations in early-passage cells from ten newly established ATC cell lines using single nucleotide polymorphism (SNP) array analysis, RNA sequencing and whole exome sequencing. The ATC cell line genomes were highly complex and displayed signs of replicative stress and genomic instability, including massive aneuploidy and frequent breakpoints in the centromeric regions and in fragile sites. Loss of heterozygosity involving whole chromosomes was common, but there were no signs of previous near-haploidisation events or chromothripsis. A total of 21 fusion genes were detected, including six predicted in-frame fusions; none were recurrent. Global gene expression analysis showed 661 genes to be differentially expressed between ATC and papillary thyroid cancer cell lines, with pathway enrichment analyses showing downregulation of TP53 signalling as well as cell adhesion molecules in ATC. Besides previously known driver events, such as mutations in BRAF, NRAS, TP53 and the TERT promoter, we identified PTPRD and NEGR1 as putative novel target genes in ATC, based on deletions in six and four cell lines, respectively; the latter gene also carried a somatic mutation in one cell line. Taken together, our data provide novel insights into the tumourigenesis of ATC and may be used to identify new therapeutic targets.

Transcriptomic comparison of primary bovine horn core carcinoma culture and parental tissue at early stage

Aim:Squamous cell carcinoma or SCC of horn in bovines (bovine horn core carcinoma) frequently observed in Bos indicus affecting almost 1% of cattle population. Freshly isolated primary epithelial cells may be closely related to the malignant epithelial cells of the tumor. Comparison of gene expression in between horn’s SCC tissue and its early passage primary culture using next generation sequencing was the aim of this study.Materials and Methods:Whole transcriptome sequencing of horn’s SCC tissue and its early passage cells using Ion Torrent PGM were done. Comparative expression and analysis of different genes and pathways related to cancer and biological processes associated with malignancy, proliferating capacity, differentiation, apoptosis, senescence, adhesion, cohesion, migration, invasion, angiogenesis, and metabolic pathways were identified.Results:Up-regulated genes in SCC of horn’s early passage cells were involved in transporter activity, catalytic activity, nucleic acid binding transcription factor activity, biogenesis, cellular processes, biological regulation and localization and the down-regulated genes mainly were involved in focal adhesion, extracellular matrix receptor interaction and spliceosome activity.Conclusion:The experiment revealed similar transcriptomic nature of horn’s SCC tissue and its early passage cells.

ITRAQ-Based Quantitative Proteomic Comparison of Early- and Late-Passage Human Dermal Papilla Cell Secretome in Relation to Inducing Hair Follicle Regeneration.

Alopecia is an exceedingly prevalent problem that lacks effective therapy. Recently, research has focused on early-passage dermal papilla cells (DPCs), which have hair inducing activity both in vivo and in vitro. Our previous study indicated that factors secreted from early-passage DPCs contribute to hair follicle (HF) regeneration. To identify which factors are responsible for HF regeneration and why late-passage DPCs lose this potential, we collected 48-h-culture medium (CM) from both of passage 3 and 9 DPCs, and subcutaneously injected the DPC-CM into NU/NU mice. Passage 3 DPC-CM induced HF regeneration, based on the emergence of a white hair coat, but passage 9 DPC-CM did not. In order to identify the key factors responsible for hair induction, CM from passage 3 and 9 DPCs was analyzed by iTRAQ-based quantitative proteomic technology. We identified 1360 proteins, of which 213 proteins were differentially expressed between CM from early-passage vs. late-passage DPCs, including SDF1, MMP3, biglycan and LTBP1. Further analysis indicated that the differentially-expressed proteins regulated the Wnt, TGF-β and BMP signaling pathways, which directly and indirectly participate in HF morphogenesis and regeneration. Subsequently, we selected 19 proteins for further verification by multiple reaction monitoring (MRM) between the two types of CM. These results indicate DPC-secreted proteins play important roles in HF regeneration, with SDF1, MMP3, biglycan, and LTBP1 being potential key inductive factors secreted by dermal papilla cells in the regeneration of hair follicles.

Co-existence of BRAF and NRAS driver mutations in the same melanoma cells results in heterogeneity of targeted therapy resistance.

Acquired chemotherapeutic resistance of cancer cells can result from a Darwinistic evolution process in which heterogeneity plays an important role. In order to understand the impact of genetic heterogeneity on acquired resistance and second line therapy selection in metastatic melanoma, we sequenced the exomes of 27 lesions which were collected from 3 metastatic melanoma patients treated with targeted or non-targeted inhibitors. Furthermore, we tested the impact of a second NRAS mutation in 7 BRAF inhibitor resistant early passage cell cultures on the selection of second line therapies.We observed a rapid monophyletic evolution of melanoma subpopulations in response to targeted therapy that was not observed in non-targeted therapy. We observed the acquisition of NRAS mutations in the BRAF mutated patient treated with a BRAF inhibitor in 1 of 5 of his post-resistant samples. In an additional cohort of 5 BRAF-inhibitor treated patients we detected 7 NRAS mutations in 18 post-resistant samples. No NRAS mutations were detected in pre-resistant samples. By sequencing 65 single cell clones we prove that NRAS mutations co-occur with BRAF mutations in single cells. The double mutated cells revealed a heterogeneous response to MEK, ERK, PI3K, AKT and multi RTK - inhibitors.We conclude that BRAF and NRAS co-mutations are not mutually exclusive. However, the sole finding of double mutated cells in a resistant tumor is not sufficient to determine follow-up therapy. In order to target the large pool of heterogeneous cells in a patient, we think combinational therapy targeting different pathways will be necessary.

Stereotactic Body Radiation Therapy Gains Radiobiological Advantages Not in Non-Small Cell Lung Cancer Cells After Radiation But in Early Passage Cells Compared to Conventional Radiation Therapy

Stereotactic Body Radiation Therapy Gains Radiobiological Advantages Not in Non-Small Cell Lung Cancer Cells After Radiation But in Early Passage Cells Compared to Conventional Radiation Therapy

P08.57 Involvement of both the extrinsic and intrinsic apoptotic pathways with clomipramine treatment of human glioblastoma cells under normoxic and hypoxic conditions

AbstractIntroduction:The tricyclic antidepressant clomipramine, used clinically since the 1960’s, has been shown to have selective cytotoxicity against glioma cells in vitro and has also been shown to cross the blood brain barrier. While reports suggest clomipramine acts via the mitochondria, the mechanism of action is not fully understood.Materials and Methods:In this study, 8 human biopsy-derived glioblastoma early passage cell lines were treated with 0–200µM clomipramine over 72hrs. Cell viability was measured using the MTS assay, cellular ATP levels using the Cell Titer Glo assay and capsase activation by Caspase-Glo 3/7, 8 and 9 assays. Immunocytochemistry using Mitotracker Red and cytochrome C antibodies was performed. The JC-1 dye was used to evaluate mitochondrial transmembrane potential and DAPI was used to characterise the cell cycle.Results:Clomipramine reduced cell viability in a dose and time dependent manner with IC50 values in the range of 22–60µM at 24hrs. Compared to controls, following 24 hr treatment with 75µM clomipramine, there was a significant reduction of cellular ATP levels (by approximately 100%, p<0.0001), depolarisation of the mitochondrial transmembrane potential and release of cytochrome C from the mitochondria. The extrinsic apoptotic pathway was activated at 12 hours whereas the intrinsic (mitochondrial-mediated) pathway was activated at 24 hours as shown by elevation of caspase 8, 9 and 3/7 levels. The reduction in cell viability in response to clomipramine was attenuated by the pan-caspase inhibitor Z-VAD-FMK, indicating involvement of caspases in the mechanism of action. With the most significant effects observed with SEBTA-023; 24 hours at 75µM clomipramine cell viability increased by 50% and 67% under hypoxic and normoxic conditions respectively (p<0.0001). The effects on the cell cycle were evaluated and clomipramine treatment led to cell cycle arrest in G2/M phase. Two other tricyclic antidepressants, imipramine and amitriptyline were also examined and clomipramine was deemed to be the most effective inducer of apoptosis.Conclusions:This work provides further insights to the mechanism of action of this class of repurposed drug as a potential adjuvant treatment for GBM.

Analysis of Body-wide Unfractionated Tissue Data to Identify a Core Human Endothelial Transcriptome

Endothelial cells line blood vessels and regulate hemostasis, inflammation, and blood pressure. Proteins critical for these specialized functions tend to be predominantly expressed in endothelial cells across vascular beds. Here, we present a systems approach to identify a panel of human endothelial-enriched genes using global, body-wide transcriptomics data from 124 tissue samples from 32 organs. We identified known and unknown endothelial-enriched gene transcripts and used antibody-based profiling to confirm expression across vascular beds. The majority of identified transcripts could be detected in cultured endothelial cells from various vascular beds, and we observed maintenance of relative expression in early passage cells. In summary, we describe a widely applicable method to determine cell-type-specific transcriptome profiles in a whole-organism context, based on differential abundance across tissues. We identify potential vascular drug targets or endothelial biomarkers and highlight candidates for functional studies to increase understanding of the endothelium in health and disease.

Abstract 843: Spontaneous immortalization of human mammary epithelial cells from a woman with a germline STK11 mutation

Abstract Spontaneous immortalization of benign mammary epithelial cells (MEC) in culture is very rare. Models of MEC immortalization suggest that cells escape M0 growth arrest (selection) by silencing p16 expression after which they encounter a second growth arrest, M1 (agonescence), characterized by telomere shortening and profound genomic instability. Pre-selection, post-selection, and spontaneously immortalized MEC from a woman with a germline STK11 mutation were subjected to a comprehensive “OMICS” evaluation including miRNA, transcriptome, methylome, exome, and comparative genomic hybridization (CGH) in order to understand the molecular events associated with MEC immortalization. Principle component analysis of gene expression identified 4 significant components, the first of which showed the greatest separation between the different time points. The most notable expression changes were upregulation of FOXQ1, IL1-related genes, Ras-related genes, and fibronectin and downregulation of COL1, COL6, CK19, and CDH1. MEC gradually acquired global hypomethylation of non-CpG DNA and regional hypermethylation of CpG islands (including p16). A classifier based on methylation pattern showed that immortalized cells moved away from identity with The Cancer Genome Atlas (TCGA) benign samples towards TCGA breast cancer samples. Immortalization was associated with a modest accumulation of mutations and a modestly greater number of DNA copy number alterations than was observed in earlier passage cells but no evidence of general genomic instability. Immortal cells showed a large deletion at 10q13-15 and deletions at 10p11-15 and 17p11-13 that were associated with reduced expression of genes in those regions. Post-selection bulk cell populations are quite distinct from early passage cells, but are not direct precursors of the immortal cells as some DNA copy number alterations acquired by post-selection cells were not observed in the immortalized cells. Rather, in this instance, it is likely that post-selection cells established conditions conducive to the immortalization of a rare cell in the mix. This has implications for the development of tissue-based approaches for breast cancer risk stratification. Citation Format: David Euhus, Dawei Bu, Michael Considine, Leslie Cope. Spontaneous immortalization of human mammary epithelial cells from a woman with a germline STK11 mutation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 843.

Modeling fibrosis using fibroblasts isolated from scarred rat vocal folds

Following injury, pathologically activated vocal fold fibroblasts (VFFs) can engage in disordered extracellular matrix (ECM) remodeling, leading to VF fibrosis and impaired voice function. Given the importance of scar VFFs to phenotypically appropriate in vitro modeling of VF fibrosis, we pursued detailed characterization of scar VFFs obtained from surgically injured rat VF mucosae, compared with those obtained from experimentally naïve, age-matched tissue. Scar VFFs initially exhibited a myofibroblast phenotype characterized by increased proliferation, increased Col1a1 transcription and collagen, type I synthesis, increased Acta2 transcription and α-smooth muscle actin synthesis, and enhanced contractile function. These features were most distinct at passage 1 (P1); we observed a coalescence of the scar and naïve VFF phenotypes at later passages. An empirical Bayes statistical analysis of the P1 cell transcriptome identified 421 genes that were differentially expressed by scar, compared with naïve, VFFs. These genes were primarily associated with the wound response, ECM regulation, and cell proliferation. Follow-up comparison of P1 scar VFFs and their in vivo tissue source showed substantial transcriptomic differences. Finally, P1 scar VFFs responded to treatment with hepatocyte growth factor and transforming growth factor-β3, two biologics with reported therapeutic value. Despite the practical limitations inherent to working with early passage cells, this experimental model is easily implemented in any suitably equipped laboratory and has the potential to improve the applicability of preclinical VF fibrosis research.

Structural and quantitative evidence of \u03b12\u20136-sialylated N-glycans as markers of the differentiation potential of human mesenchymal stem cells

Human somatic stem cells such as mesenchymal stem cells (hMSCs) have the capacity to differentiate into mesenchymal tissue lineages and to alter immune regulatory functions. As such, they hold promise for use in stem cell-based therapies. However, no method is currently available to evaluate the actual differentiation capacity of hMSCs prior to cell transplantation. Previously, we performed a comprehensive glycan profiling of adipose-derived hMSCs using high-density lectin microarray and demonstrated that α2–6-sialylation is a marker of the differentiation potential of these cells. Nevertheless, no information was available about the structural details of these of α2–6-sialylated glycans. Here we used high performance liquid chromatography (HPLC) analysis combined with mass spectrometry (MS) to perform a structural and quantitative glycome analysis targeting both N- and O-glycans derived from early (with differentiation ability) and late (without differentiation ability) passages of adipose tissue-derived hMSCs. Findings in these cells were compared with those from human induced pluripotent stem cells (hiPSCs), human dermal fibroblasts (hFibs) and cartilage tissue-derived chondrocytes. A higher percentage of α2–6-sialylated N-glycans was detected in early passage cells (24–28 % of sialylated N-glycans) compared with late passage cells (13–15 %). A major α2–6-sialylated N-glycan structure detected in adipose-derived hMSCs was that of mono-sialylated biantennary N-glycan. Similar results were obtained for the cartilage tissue-derived chondrocytes, Yub621c (28 % for passage 7 and 5 % for passage 28). In contrast, no significant differences were observed between early and late passage hMSCs with respect to α2–6-sialylated O-glycan percentages. These results demonstrate that levels of α2–6-sialylated N-glycans, but not O-glycans, could be used as markers of the differential potential of hMSCs.

Glucocorticoid Cell Priming Enhances Transfection Outcomes in Adult Human Mesenchymal Stem Cells.

Human mesenchymal stem cells (hMSCs) are one of the most widely researched stem cell types with broad applications from basic research to therapeutics, the majority of which require introduction of exogenous DNA. However, safety and scalability issues hinder viral delivery, while poor efficiency hinders nonviral gene delivery, particularly to hMSCs. Here, we present the use of a pharmacologic agent (glucocorticoid) to overcome barriers to hMSC DNA transfer to enhance transfection using three common nonviral vectors. Glucocorticoid priming significantly enhances transfection in hMSCs, demonstrated by a 3-fold increase in efficiency, 4-15-fold increase in transgene expression, and prolonged transgene expression when compared to transfection without glucocorticoids. These effects are dependent on glucocorticoid receptor binding and caused in part by maintenance of normal metabolic function and increased cellular (5-fold) and nuclear (6-10-fold) DNA uptake over hMSCs transfected without glucocorticoids. Results were consistent across five human donors and in cells up to passage five. Glucocorticoid cell priming is a simple and effective technique to significantly enhance nonviral transfection of hMSCs that should enhance their clinical use, accelerate new research, and decrease reliance on early passage cells.

Novel Immortal Cell Lines Support Cellular Heterogeneity in the Human Annulus Fibrosus.

IntroductionLoss of annulus fibrosus (AF) integrity predisposes to disc herniation and is associated with IVD degeneration. Successful implementation of biomedical intervention therapy requires in-depth knowledge of IVD cell biology. We recently generated unique clonal human nucleus pulposus (NP) cell lines. Recurring functional cellular phenotypes from independent donors provided pivotal evidence for cell heterogeneity in the mature human NP. In this study we aimed to generate and characterize immortal cell lines for the human AF from matched donors.MethodsNon-degenerate healthy disc material was obtained as surplus surgical material. AF cells were immortalized by simian virus Large T antigen (SV40LTAg) and human telomerase (hTERT) expression. Early passage cells and immortalized cell clones were characterized based on marker gene expression under standardized culturing and in the presence of Transforming Growth factor β (TGFβ).ResultsThe AF-specific expression signature included COL1A1, COL5A1, COL12A1, SFRP2 and was largely maintained in immortal AF cell lines. Remarkably, TGFβ induced rapid 3D sheet formation in a subgroup of AF clones. This phenotype was associated with inherent differences in Procollagen type I processing and maturation, and correlated with differential mRNA expression of Prolyl 4-hydroxylase alpha polypeptide 1 and 3 (P4HA1,3) and Lysyl oxidase (LOX) between clones and differential P4HA3 protein expression between AF cells in histological sections.ConclusionWe report for the first time the generation of representative human AF cell lines. Gene expression profile analysis and functional comparison of AF clones revealed variation between immortalized cells and suggests phenotypic heterogeneity in the human AF. Future characterization of AF cellular (sub-)populations aims to combine identification of additional specific AF marker genes and their biological relevance. Ultimately this knowledge will contribute to clinical application of cell-based technology in IVD repair.

The Function and Molecular Mechanism of Cellular Senescence-Inhibited Gene

To explore the genetic mechanism of replicative senescence, we identified and cloned a Cellular Senescence-Inhibited Gene/ribosomal L1 domain containing 1 (CSIG/RSL1D1) through suppressive subtractive hybridization. CSIG expression is high in earlypassaged human diploid fibroblast cells but decreased during senescence. Our results demonstrated that CSIG inhibited the senescence progression and mediated the apoptosis signaling pathway under ultraviolet (UV) irradiation. Moreover, emerging evidences have indicated that CSIG is involved in other processes including cell cycle regulation and tumor development. This is a brief overview to introduce the studies on the function and molecular mechanism of CSIG

Simultaneous Multiplexed Measurement of RNA and Proteins in Single Cells.

SummarySignificant advances have been made in methods to analyze genomes and transcriptomes of single cells, but to fully define cell states, proteins must also be accessed as central actors defining a cell’s phenotype. Methods currently used to analyze endogenous protein expression in single cells are limited in specificity, throughput, or multiplex capability. Here, we present an approach to simultaneously and specifically interrogate large sets of protein and RNA targets in lysates from individual cells, enabling investigations of cell functions and responses. We applied our method to investigate the effects of BMP4, an experimental therapeutic agent, on early-passage glioblastoma cell cultures. We uncovered significant heterogeneity in responses to treatment at levels of RNA and protein, with a subset of cells reacting in a distinct manner to BMP4. Moreover, we found overall poor correlation between protein and RNA at the level of single cells, with proteins more accurately defining responses to treatment.

Evaluating the Effect of Cell Culture on Gene Expression in Primary Tissue Samples Using Microfluidic-Based Single Cell Transcriptional Analysis.

Significant transcriptional heterogeneity is an inherent property of complex tissues such as tumors and healing wounds. Traditional methods of high-throughput analysis rely on pooling gene expression data from hundreds of thousands of cells and reporting a population-wide average that is unable to capture differences within distinct cell subsets. Recent advances in microfluidic technology have permitted the development of large-scale single cell analytic methods that overcome this limitation. The increased granularity afforded by such approaches allows us to answer the critical question of whether expansion in cell culture significantly alters the transcriptional characteristics of cells isolated from primary tissue. Here we examine an established population of human adipose-derived stem cells (ASCs) using a novel, microfluidic-based method for high-throughput transcriptional interrogation, coupled with advanced bioinformatic analysis, to evaluate the dynamics of single cell gene expression among primary, passage 0, and passage 1 stem cells. We find significant differences in the transcriptional profiles of cells from each group, as well as a considerable shift in subpopulation dynamics as those subgroups better able to adhere and proliferate under these culture conditions gradually emerge as dominant. Taken together, these findings reinforce the importance of using primary or very early passage cells in future studies.

Inhibitory effects of PPARγ ligands on TGF-β1-induced CTGF expression in cat corneal fibroblasts

Ligands of Peroxisome Proliferator Activated Receptor gamma (PPARγ) possess strong anti-fibrotic properties in the cornea and several other body tissues. In the cornea, we recently showed this class of molecules to prevent stromal myofibroblast differentiation partially by blocking the actions of p38 mitogen-activated protein kinase (MAPK). However, given the important role assigned to connective tissue growth factor (CTGF) in mediating corneal fibrosis, here we asked whether PPARγ ligands also act by affecting transforming growth factor-β (TGF-β) 1-induced expression of CTGF in cultured corneal fibroblasts. Corneal keratocytes were isolated from young, adult cats and early passage cells were exposed to TGF-β1 with or without the PPARγ ligands Rosiglitazone, Troglitazone and 15d-PGJ2. Western blots were used to assay levels of CTGF and alpha smooth muscle actin (αSMA), a marker of myofibroblast differentiation. CTGF siRNA demonstrated a critical role for CTGF in TGF-β1-mediated myofibroblast differentiation, while exogenously applied CTGF potentiated the pro-fibrogenic effects of TGF-β1. TGF-β1-mediated increases in CTGF and αSMA expression were strongly inhibited by all three PPARγ ligands tested, and by a c-jun N-terminal kinase (JNK) inhibitor. However, while extracellular signal-regulated kinase (ERK) 1/2, protein kinase B (AKT) and p38 MAPK inhibitors also blocked TGF-β1-induced αSMA induction, they did not dampen TGF-β1-induced increases in levels of CTGF. Thus, we conclude that PPARγ ligands block TGF-β1-induced increases in CTGF levels in cat corneal fibroblasts. They appear to do this in addition to their anti-fibrotic effect on p38 MAPK, providing a second intracellular pathway by which PPARγ ligands block αSMA induction.