Early Passage Cells Research Articles

Joint action of miR-126 and MAPK/PI3K inhibitors against metastatic melanoma.

Emerging data support the rationale of combined therapies in advanced melanoma. Specifically, the combined use of drugs with different mechanisms of action can reduce the probability of selecting resistant clones. To identify agents active against melanoma cells, we screened a library of 349 anti‐cancer compounds, currently in clinical use or trials, and selected PIK‐75, an inhibitor of the phosphatidylinositol 3‐kinase/protein kinase B (PI3K/AKT) pathway, as the ‘top active’ drug. PIK‐75 was then used alone or in combination with vemurafenib, the first BRAF inhibitor approved for patients with melanoma harboring BRAF mutations. We identified a combined dose of PIK‐75 and vemurafenib that inhibited both the PI3K/AKT and mitogen‐activated protein kinase pathways, thereby overcoming any compensatory activation. In view of the important tumor suppressor function induced by restoring expression of microRNA (miR)‐126 in metastatic melanoma cells, we examined whether miR‐126 has a synergistic role when included in a triple combination alongside PIK‐75 and vemurafenib. We found that enforced expression of miR‐126 (which alone can reduce tumorigenicity) significantly increased PIK‐75 activity when used as either a single agent or in combination with vemurafenib. Interestingly, PIK‐75 proved to be effective against early passage cell lines derived from patients’ biopsies and on melanoma cell lines resistant to either vemurafenib or dabrafenib, thus suggesting that it potentially has the capability to overcome drug resistance. Finally, the synergistic role played by miR‐126 in combination with vemurafenib and/or PIK‐75 was demonstrated in vivo in mouse xenograft models, in which tumor growth inhibition was associated with increased apoptosis. These results not only show the efficacy of PIK‐75 and vemurafenib co‐treatment but also indicate that restoration of miR‐126 expression in advanced melanoma can enhance their antitumor activity, which may possibly allow dose reduction to decrease adverse events without reducing the therapeutic benefits.

Ovarian Cancer Treatment Stratification Using Ex Vivo Drug Sensitivity Testing.

Treatment options for patients with platinum-resistant ovarian cancer are generally palliative in nature and rarely have realistic potential to be curative. Because many patients with recurrent ovarian cancer receive aggressive chemotherapy for prolonged periods, sometimes continuously, therapy-related toxicities are a major factor in treatment decisions. The use of ex vivo drug sensitivity screens has the potential to improve the treatment of patients with platinum-resistant ovarian cancer by providing personalized treatment plans and thus reducing toxicity from unproductive therapy attempts. We evaluated the treatment responses of a set of six early-passage patient-derived ovarian cancer cell lines towards a set of 30 Food and Drug Administration-approved chemotherapy drugs using drug-sensitivity testing. We observed a wide range of treatment responses of the cell lines. While most compounds displayed vastly different treatment responses between cell lines, we found that some compounds such as docetaxel and cephalomannine reduced cell survival of all cell lines. We propose that ex vivo drug-sensitivity screening holds the potential to greatly improve patient outcomes, especially in a population where multiple continuous treatments are not an option due to advanced disease, rapid disease progression, age or poor overall health. This approach may also be useful to identify potential novel therapeutics for patients with ovarian cancer.

Qualifying mscs for clinical applications -differential gene expression analyses of pluripotent, differentiation and surface marker genes of early and late passage mscs, in hypoxic and normal oxygen level in culture conditions.

Background & Aim With the increasing focus on the large scale expansion of MSCs required for the clinical application, the optimisation of in-vitro expansion of MSCs requires careful consideration to maintain the native MSCs characteristics. Physiological parameters such as oxygen concentration, media constituents and passage numbers have much influence on the properties of MSCs and may have major impact on the therapeutic potential of MSCs. Cells grown under hypoxic (low oxygen) conditions have been widely documented in clinical use however culturing MSCS in large scale requires bioreactor and it is challenging to maintain the low oxygen and other physiological parameters over several passages in big vessels of bioreactors. Similarly, the culture conditions in the bioreactor including pH, dissolved oxygen and the sheer force exerted on cells may change the cellular characteristics including gene expression Methods, Results & Conclusion These culture conditions may have different potency attributes clinically. For these reasons, our study investigated differential expression of 11 key genes of MSCs for pluripotent genes POU5F1, NANOG and KLF4, differentiation genes RUNX2, COL1A1, SOX9, COL2A1, PPARG and CD marker genes THY1, ENG and NT5E from adipose derived MSCs, grown under both the normal oxygen or hypoxic conditions were analysed. Two different cells age early (P5) and late passage (P9) cells. The result demonstrated marked differences in gene expression pattern of the early and late passage cells grown under normoxic (normal oxygen) when compared with the hypoxic conditions. Pluripotent markers KLF4 in early passage cells was expressed more than two-fold in the normoxic cells, and PPARG was expressed more in the late passage cells under normoxic condition. Differentiation marker for RUNX2 and NT5E were highly expressed in early passage cells. These differences in gene expression may have critical impact on the potency of the cells grown in large scale in bioreactor. Additionally, the effects of prolonged culture of MSCs in-vitro will need be qualified prior to use when manufactured in large scale bioreactor for the clinical dose to ensure the therapeutic efficacy.

The development and characterization of patient derived xenografts from non-small cell lung cancer brain metastases

Introduction: NSCLC brain metastasis cell lines and in vivo brain metastasis models are not widely accessible. The purpose of this study was to establish and characterize a direct-from patient-derived xenograft (PDX) model of non-small cell lung cancer (NSCLC) brain metastases. Methods: Surgically obtained tissue was implanted subcutaneously and as orthotopic intracranial implants into immunodeficient mice. Histology and DNA loci were compared between original tumor and subsequent PDX passages. Tumors underwent RNA and DNA sequencing and relevant therapeutic targets were identified. Tumor growth rates were assessed following treatment with radiation, MEK inhibitor selumetinib, or MET inhibitor savolitinib. Cell lines were established. Results: Nine NSCLC brain metastases PDXs were established. Morphologically, strong retention of cytoarchitectural features was observed between original patient tumor and subcutaneous and intracranial tumors. Preservation of thyroid transcription factor 1 expression was seen in all xenografts. Short tandem repeat analysis demonstrated strong concordance between patient tumors and subsequent PDX passages. RNA sequencing analysis revealed high correlation between matched patient and PDX samples. Significant growth inhibition was shown with radiation, with selumetinib in tumors harboring KRAS G12C mutation and with savolitinib in tumors with MET exon 14 skipping mutation. Significant tumor growth delay was observed with the combination of radiation and savolitinib compared to radiation or savolitinib alone in the PDX harboring MET exon 14 skipping mutation. Early passage cell strains showed high consistency between patient and PDX tumors. Conclusion: We have established a robust human xenograft model system for investigating NSCLC brain metastases. These PDXs and cell lines show strong phenotypic and molecular correlation with the original patient tumors and provide a valuable resource for testing preclinical therapeutics.

Changes in SOX9 and RUNX2 protein activity correlate to the health state of human primary chondrocytes

Purpose: SOX9 and RUNX2 are the master transcription factors involved in cartilage and bone development respectively. Altered SOX9 and RUNX2 activity is implicated in osteoarthritis (OA) pathophysiology and hypertrophic differentiation of chondrocytes. We have previously shown that SOX9 and RUNX2 protein activity can be measured by Transcription Factor - Fluorescence Recovery After Photobleaching (TF-FRAP). To decipher the underlying signaling mechanism in OA pathophysiology, we aim to use TF-FRAP to correlate SOX9 and RUNX2 mobility to the health state of the chondrocytes. We hypothesize that the mobility of SOX9 increases with OA progression, while the mobility of RUNX2 decreases in chondrocyte hypertrophy. We measured the mobility and correlated protein activity of these transcription factors at the single cell level by TF-FRAP in healthy, preserved and OA chondrocytes. Methods: Healthy human primary chondrocytes (hPCs) were purchased from Articular Engineering, USA. Human preserved and OA hPCs were isolated from patients undergoing total knee replacement therapy. hPCs were seeded on glass coverslips and transfected with either SOX9-mGFP or eGFP-RUNX2. TF-FRAP was performed in at least 40 cells per condition in two healthy donors and three donors for each preserved and OA chondrocytes. TF-FRAP measurements were done using a Nikon A1 confocal microscope (Japan). In TF-FRAP, a small circular region (Ø 25 pixels) of the nucleus was bleached using a high intensity laser and the recovery of the fluorescence in the bleach spot is recorded. For every TF-FRAP measurement, 25 pre-bleach images and 260 post-bleach images were acquired at 4 frames per second for 60 seconds, with a frame size of 256x256 pixels. Higher immobile fraction (i.e., the fraction of transcription factors bound to DNA) and higher half-time recovery (longer DNA interaction times) were associated with a higher transcriptional activity. For all donors, early passage (≤3) cells were used for TF-FRAP and qPCR measurements. To correlate protein mobility and DNA binding with the protein activity, we quantified the expression levels of SOX9 target genes (ACAN and COL2A) and RUNX2 target genes (MMP13 and COL10) by qPCR. Results: In the healthy hPCs, the SOX9 immobile fraction and half-time recovery were 60% (SD ±6%) and 14.2 sec (SD ±3.2 sec) respectively. In preserved hPCs, the SOX9 immobile fraction and half-time to recover was significantly lower, ranging from 50% - 44% (SD ±7% - ±9.7%), and 14.2 - 13.3 sec (SD ±3.9 - ±4.5 sec) respectively. In OA hPCs, the SOX9 immobile fraction and half-time recovery was significantly lower than in the healthy and preserved hPCs, ranging from 48.6% - 42.8% (SD ±7.3% - ±8.4%), and half-time to recover ranged from 13.3 - 12.2 sec (SD ±3.9 - ±5.3 sec). RUNX2 immobile fraction and half-time to recover data spread was large in healthy, preserved and OA hPCs. In healthy hPCs, immobile fraction and half-time to recover were ranging from 33.6% - 47.9% (SD 19.4% - 20.7%) and 13.97 sec - 14.46 sec (SD 7.9 - 10.1 sec) respectively. In the preserved hPCs, the immobile fraction and half-time to recover were ranging from 38.8% - 47.0 % (SD 19.7% - 22.2%) and 11.7 sec - 14.8 sec (SD 6.8 - 8.2 sec) respectively. In OA hPCs, the immobile fraction and half-time to recover ranged from 33.0% - 41.02% (SD 20.3% - 25.7%) and 11.2 sec - 12.9 sec (SD 7.4 - 9.1 sec) respectively. We have an initial non-quantitative indication for a distinct cell population that, irrespective of the health state, shows decreased RUNX2 mobility, which could be indicative of a hypertrophic cell state. We will show the quantification and segregation. RT-qPCR measurements show decreased SOX9 and increased RUNX2 target gene expression during OA progression. Conclusions: Our data show that the SOX9 and RUNX2 transcriptional activity is differentially regulated depending on the health state of hPCs. If we assume a correlation between mobility and transcription factor activity, then we can say that SOX9 protein activity decreases with increasing OA progression. RUNX2 mobility data showed large spread especially in the preserved and OA donors possibly due to cells were in the different health state in the OA joint. Our findings at the single cell level provide novel insights into SOX9 and RUNX2 protein activity with respect to OA progression.

Accumulating Transcriptome Drift Precedes Cell Aging in Human Umbilical Cord-Derived Mesenchymal Stromal Cells Serially Cultured to Replicative Senescence.

In preclinical studies, mesenchymal stromal cells (MSCs) exhibit robust potential for numerous applications. To capitalize on these benefits, cell manufacturing and delivery protocols have been scaled up to facilitate clinical trials without adequately addressing the impact of these processes on cell utility nor inevitable regulatory requirements for consistency. Growing evidence indicates that culture-aged MSCs, expanded to the limits of replicative exhaustion to generate human doses, are not equivalent to early passage cells, and their use may underpin reportedly underwhelming or inconsistent clinical outcomes. Here, we sought to define the maximum expansion boundaries for human umbilical cord-derived MSCs, cultured in chemically defined xeno- and serum-free media, that yield consistent cell batches comparable to early passage cells. Two male and two female donor populations, recovered from cryostorage at mean population doubling level (mPDL) 10, were serially cultivated until replicative exhaustion (senescence). At each passage, growth kinetics, cell morphology, and transcriptome profiles were analyzed. All MSC populations displayed comparable growth trajectories through passage 9 (P9; mPDL 45) and variably approached senescence after P10 (mPDL 49). Transcription profiles of 14,500 human genes, generated by microarray, revealed a nonlinear evolution of culture-adapted MSCs. Significant expression changes occurred only after P5 (mPDL 27) and accumulated rapidly after P9 (mPDL 45), preceding other cell aging metrics. We report that cryobanked umbilical cord-derived MSCs can be reliably expanded to clinical human doses by P4 (mPDL 23), before significant transcriptome drift, and thus represent a mesenchymal cell source suited for clinical translation of cellular therapies. Stem Cells Translational Medicine 2019;8:945&958.

Glia-Like Cells From Late-Passage Human MSCs Protect Against Ischemic Stroke Through IGFBP-4

Summary: Early-passage human mesenchymal stem cells (hMSCs) are typically used in clinical trials because of safety and efficacy issues. However, obtaining sufficient cells for treatment is difficult and expensive. Many more late-passage hMSCs can be obtained at lower cost, although efficacy is a large hurdle in clinical trials. The use of late-passage MSCs with better efficacy would be a revolutionary solution for reducing cost and facilitating clinical trials. In the present study, glia-like cells (ghMSCs) were induced from hMSCs and used for both in vitro and in vivo models of ischemia, effectively protecting neurons from ischemia and restoring brain tissue damaged by cerebral infarction. These beneficial effects were significantly blocked by insulin-like growth factor-binding protein-4 (IGFBP-4) antibody. The current study demonstrated that late-passage hMSCs can be efficiently induced into ghMSCs with a better neuroprotective effect against ischemic stroke and that IGFBP-4 may be a key neuronal survival factor secreted by ghMSCs. Funding Statement: This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI17C2160). Declaration of Interests: One of the corresponding authors, Mi-Sook Chang, has a patent related to this work, which is noted in the Declaration of Interests section of the manuscript. She obtained a Korean patent for glia-like human mesenchymal stem cells in 2012. Ethics Approval Statements: All procedures involving animals were approved by the Institutional Animal Care and Use Committee, Seoul National University (Seoul, Republic of Korea). In addition, all animal procedures were performed in accordance with the Hanyang University guidelines for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee (IACUC) of Hanyang University. In accordance with Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines, all experiments were carried out in a strictly blinded fashion, inclusion and exclusion criteria were predetermined, and attrition due to mortality and other causes was reported

Impaired proteostasis in senescent vascular endothelial cells: a perspective on estrogen and oxidative stress in the aging vasculature.

The heat shock response is an important cytoprotective mechanism for protein homeostasis and is an essential protective response to cellular stress and injury. Studies on changes in the heat shock response with aging have been mixed with regard to whether it is inhibited, and this, at least in part, reflects different tissues and different models. Cellular senescence is a key feature in aging, but work on the heat shock response in cultured senescent (SEN) cells has largely been limited to fibroblasts. Given the prevalence of oxidative injury in the aging cardiovascular system, we investigated whether SEN primary human coronary artery endothelial cells have a diminished heat shock response and impaired proteostasis. In addition, we tested whether this downregulation of heat shock response can be mitigated by 17β-estradiol (E2), which has a critical cardioprotective role in women, as we have previously reported that E2 improves the heat shock response in endothelial cells (Hamilton KL, Mbai FN, Gupta S, Knowlton AA. Arterioscler Thromb Vasc Biol 24: 1628-1633, 2004). We found that SEN endothelial cells, despite their unexpectedly increased proteasome activity, had a diminished heat shock response and had more protein aggregation than early passage cells. SEN cells had increased oxidative stress, which promoted protein aggregation. E2 treatment did not decrease protein aggregation or improve the heat shock response in either early passage or SEN cells. In summary, cellular senescence in adult human endothelial cells is accompanied by increased oxidative stress and a blunting of proteostasis, and E2 did not mitigate these changes. NEW & NOTEWORTHY Senescent human endothelial cells have a diminished heat shock response and increased protein aggregates. Senescent human endothelial cells have increased basal oxidative stress, which increases protein aggregates. Physiological level of 17β-estradiol did not improve proteostasis in endothelial cells.

Metabolic profile and differentiation potential of extraembryonic endoderm-like cells

Glucose metabolism has a crucial role for providing substrates required to generate ATP and regulate the epigenetic landscape. We reported that F9 embryonal carcinoma stem-like cells require cytosolic reactive oxygen species to differentiate into extraembryonic endoderm; however, mitochondrial sources were not examined. To extend these studies, we examined the metabolic profile of early and late-passage F9 cells, and show that their ability to differentiate is similar, even though each population has dramatically different metabolic profiles. Differentiated early-passage cells relied on glycolysis, while differentiated late-passage cells transitioned towards oxidative phosphorylation (OXPHOS). Unexpectedly, electron transport chain protein stoichiometry was disrupted in differentiated late-passage cells, whereas genes encoding mitofusion 1 and 2, which promote mitochondrial fusion and favor OXPHOS, were upregulated in differentiated early-passage cells. Despite this, early-passage cells cultured under conditions to promote glycolysis showed enhanced differentiation, whereas promoting OXPHOS in late-passage cells showed a similar trend. Further analysis revealed that the distinct metabolic profiles seen between the two populations is largely associated with changes in genomic integrity, linking metabolism to passage number. Together, these results indicate that passaging has no effect on the potential for F9 cells to differentiate into extraembryonic endoderm; however, it does impact their metabolic profile. Thus, it is imperative to determine the molecular and metabolic status of a stem cell population before considering its utility as a therapeutic tool for regenerative medicine.

Proteomic phenotyping of metastatic melanoma reveals putative signatures of MEK inhibitor response and prognosis

BackgroundGenotyping of melanomas is used to identify patients for treatment with BRAF and MEK inhibitors, but clinical responses are highly variable. This study investigated the utility of protein expression phenotyping to provide an integrated assessment of gene expression programs in BRAF/NRAS melanoma which would be useful for prognosis and may predict response to MEK inhibition.MethodsMass spectrometry profiling of early passage cell lines established from Stage III cutaneous melanomas was conducted. Basal protein expression was correlated with in vitro response to the MEK inhibitor, selumetinib. Protein expression in a cohort of 32 drug naïve BRAF/NRAS metastatic melanoma specimens was examined. The prognostic utility of a subset of these proteins and mRNA transcripts from a separate cohort was determined.ResultsUnsupervised analysis of basal cell line protein abundances delineated response to selumetinib, but BRAF/NRAS genotype did not. Resistance was associated with functions including cell motility, cell adhesion and cytoskeletal organization. Several of these response biomarkers were observed in lymph node biospecimens and correlated with melanoma-specific survival. Loss of ICAM-1 protein and mRNA expression was a strong prognosticator of diminished survival in BRAF/NRAS mutant melanoma.ConclusionsThese results demonstrate the utility of proteomic phenotyping to identify both putative biomarkers of response to MEK inhibition and prognostication associated with metastatic melanoma.

Abstract 986: The National Cancer Institute's patient-derived models repository (PDMR)

Abstract The National Cancer Institute (NCI) has developed a Patient-Derived Models Repository (PDMR) comprised of quality-controlled, early-passage, clinically-annotated patient-derived xenografts (PDXs) to serve as a resource for public-private partnerships and academic drug discovery efforts. These models are offered to the extramural community for research use (https://pdmr.cancer.gov/), along with clinical annotation and molecular information (whole exome sequence, RNASeq), which is available in a publicly accessible database. The PDMR was established by NCI at the Frederick National Laboratory for Cancer Research (FNLCR) in direct response to discussions with academia and industry; the oncology community's highest priority need was preclinical models that more faithfully reflect the patient's tumor and are associated with the patient's treatment history. NCI has focused on generating models to complement existing PDX collections and address unmet needs in the preclinical model space. The PDMR generates the majority of its PDXs by subcutaneous implantation except for those histologies having better success rates in either orthotopic or alternate implant sites. All SOPs and quality-control standards developed by the PDMR as well as those shared by collaborators are posted to a public web site that houses the PDMR database. In May 2017, the public website (https://pdmr.cancer.gov/) went live with its first 100 models from histologies including pancreatic, colorectal, renal, head and neck, and lung squamous cell cancers as well as melanoma and adult soft tissue sarcomas. In early 2018, the PDMR will begin releasing models from gynecological cancers, small cell lung cancer, chondro/osteo sarcomas, lung adenocarcinoma, and squamous cell skin and Merkel cell carcinomas. In addition, wherever available germline sequence and somatic variant calls will be added to the existing molecular characterization data for each model. NCI has also increased its focus on creating PDXs from racial and ethnic minorities through several funding opportunities. The overall goal of NCI is to create a long-term home for at least 1000 models such that sufficient biological and clinical diversity is represented to allow researchers to ask questions regarding the impact of tumor heterogeneity on target qualification or clinical response, whether PDXs more faithfully represent the human tumor for pharmacodynamic assay and predictive marker development, or if adequately powered preclinical PDX clinical trials can lead to better evaluation of therapies for future clinical use. Moving forward the PDMR plans to distribute in vitro, early-passage tumor cell cultures and cancer-associated fibroblasts as well as releasing PDX drug response data for a panel of FNA-approved therapeutic agents. Funded by NCI Contract No. HHSN261200800001E Citation Format: Yvonne A. Evrard, Michelle M. Gottholm Ahalt, Sergio . Y. Alcoser, Kaitlyn Arthur, Mariah Baldwin, Linda L. Blumenauer, Carrie Bonomi, Suzanne Borgel, Elizabeth Bradtke, Corinne Camalier, John Carter, Tiffanie Chase, Alice Chen, Lily Chen, Donna W. Coakley, Nicole E. Craig, Biswajit Das, Vivekananda Datta, Jordyn Davidson, Margaret R. DeFreytas, Emily Delaney, Michelle A. Eugeni, Raymond Divelbiss, Palmer Fliss, Thomas Forbes, Marion Gibson, Tara Grinnage-Pulley, Sierra Hoffman, Lilia Ileva, Paula Jacobs, Franklyn Jimenez, Joseph Kalen, Catherine Karangwa, Chris Karlovich, Candace Mallow, Chelsea McGlynn, Jenna E. Moyer, Michael Mullendore, Dianne L. Newton, Nimit Patel, Rajesh Patidar, Kevin Plater, Marianne Radzyminski, Lisa Riffle, Larry Rubinstein, Luke H. Stockwin, Mickey Williams, Melinda G. Hollingshead, James H. Doroshow. The National Cancer Institute's patient-derived models repository (PDMR) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 986.

Abstract 688: Differential Matricellular Protein (CCN) Expression in Atherosclerotic and Aneurysmal Smooth Muscle Cells

Introduction: Recent evidence implicates reduced activity of CCN3 as a mediator of MMP production and aortic wall degeneration in the process of model aortic aneurysm. We have previously shown that AAA-derived VSMC have increased elastolytic activity. We hypothesized that there would be differential CCN expression and production by VSMC derived from human AAA or derived from human atherosclerotic plaque. Results: Early passage cell lines of VSMC lineage as confirmed by flow cytometry were analyzed by Illumina expression array. The VSMC were derived from infrarenal aneurysm (AAA, n=22) or atherosclerotic plaque (Pl, n=29). There were no differences in expression of CCN1, 2, 5 or 6 (Fig A) between the two cell types. Expression of CCN3 was significantly reduced in AAA compared to Pl, CCN4 was significantly greater in AAA than in Pl-derived VSMC. Western blot of cellular extracts from these cells (n=3 each) confirms qualitatively greater CCN3 in Pl vs AAA VSMC, while no appreciable difference was seen in the production of CCN4 (Fig B). Production of CCN1, 2 and 6 are qualitatively similar and there may be slightly lower CCN5 production in Pl vs AAA-derived VSMC (Data not shown). Matricellular protein expression show distinct patterns between Plaque-derived and AAA-derived VSMC and may be related to the enhanced elastolytic activity of cells in AAA.

Canine corneal epithelial cells possess a sustained proliferative capacity and generate a spontaneously derived cell line

We have previously reported characteristics of canine corneal epithelial cells in vitro and found that canine corneal epithelial cells could maintain their proliferative capacity even after continuous culture without the use of feeder cells and growth promoting additives. The objective of this study was to elucidate proliferative characteristics of canine corneal epithelial cells independent of feeder cells and growth promoting additives, with the aim of developing a spontaneously derived corneal epithelial cell line. Canine and rabbit corneal epithelial cells were harvested from the limbus and cultured with, or without, feeder cells and growth promoting additives, and both were passaged continuously until growth arrest. Canine corneal epithelial cells could proliferate independently, and could be passaged more times than rabbit cells. A canine corneal epithelial cell line, cCEpi, which could be passaged more than 100 times without using feeder cells and growth promoting additives, was established. cCEpi cells maintained a cell morphology close to the primary culture and expressed p63, cytokeratin 15 (K15), and K3. Although changes in colony morphology, shortening of the population doubling time and a heteroploid karyotype were observed, cCEpi was not tumorigenic. Stratified cell sheets cultured from cCEpi were morphologically and immunohistologically similar to sheets cultivated from early passage cells. In conclusion, canine corneal epithelial cells can proliferate independent of feeder cells and growth promoting additives. cCEpi maintains properties similar to normal corneal epithelial cells and could be a useful source for studies in cellular biology and for developing novel therapies.

Automated image analysis detects aging in clinical-grade mesenchymal stromal cell cultures

BackgroundSenescent cells are undesirable in cell therapy products due to reduced therapeutic activity and risk of aberrant cellular effects, and methods for assessing senescence are needed. Early-passage mesenchymal stromal cells (MSCs) are known to be small and spindle-shaped but become enlarged upon cell aging. Indeed, cell morphology is routinely evaluated during MSC production using subjective methods. We have therefore explored the possibility of utilizing automated imaging-based analysis of cell morphology in clinical cell manufacturing.MethodsAn imaging system was adopted for analyzing changes in cell morphology of bone marrow-derived MSCs during long-term culture. Cells taken from the cultures at the desired passages were plated at low density for imaging, representing morphological changes observed in the clinical-grade cultures. The manifestations of aging and onset of senescence were monitored by population doubling numbers, expression of p16INK4a and p21Cip1/Waf1, β-galactosidase activity, and telomeric terminal restriction fragment analysis.ResultsCell area was the most statistically significant and practical parameter for describing morphological changes, correlating with biochemical senescence markers. MSCs from passages 1 (p1) and 3 (p3) were remarkably uniform in size, with cell areas between 1800 and 2500 μm2. At p5 the cells began to enlarge resulting in a 4.8-fold increase at p6–9 as compared to p1. The expression of p16INK4a and activity of β-galactosidase had a strong correlation with the increase in cell area, whereas the expression of p21Cip1/Waf1 reached its maximum at the onset of growth arrest and subsequently decreased. Mean telomere length shortened at an apparently constant rate during culture, from 8.2 ± 0.3 kbp at p1, reaching 6.08 ± 0.6 kbp at senescence.ConclusionsImaging analysis of cell morphology is a useful tool for evaluating aging in cell cultures throughout the lifespan of MSCs. Our findings suggest that imaging analysis can reproducibly detect aging-related changes in cell morphology in MSC cultures. These findings suggest that cell morphology is still a supreme measure of cell quality and may be utilized to develop new noninvasive imaging-based methods to screen and quantitate aging in clinical-grade cell cultures.

Altered properties of feline adipose-derived mesenchymal stem cells during continuous in vitro cultivation.

Cytotherapy with mesenchymal stem cells (MSCs) has been studied in many species, and often requires in vitro cell expansion to obtain therapeutic doses of stem cells. Because the characteristics of MSCs, such as self-renewal and multi-lineage differentiation, can be altered by long-term culture, it is important to maintain stemness during cultivation. This study assessed the changes in the characteristics of feline adipose tissue-derived (fAT)-MSCs during in vitro passaging. Stem cells isolated from the adipose tissue of donor cats were cultured for seven sub-passages. Proliferation capacity was analyzed by calculating the cell doubling time and by colorimetric assay. Expression of stem cell-specific markers was evaluated by quantitative reverse transcription (qRT)-PCR and immunophenotyping. Expression of adipogenic and osteogenic differentiation markers was also measured by qRT-PCR. Histochemical staining and measurement of β-galactosidase activity were conducted to detect cellular senescence. The cell proliferation rate decreased significantly at passage 5 (P5). Gene expression levels of pluripotency markers (Sox2, Nanog and Klf4) and stem cell surface markers (CD9, CD44, CD90 and CD105) decreased during continuous culture; in most assays, statistically significant changes were observed at P5. The ability of cells to undergo adipogenic or osteogenic differentiation was inversely proportional to the number of passages. The proportion of senescent cells increased with the number of passages. These results suggest that repeated passages alter the proliferation and multipotency of fAT-MSCs. In clinical trials, early-passage cells should be used to achieve the maximum therapeutic effect.

MiR-195-5p Regulates Hair Follicle Inductivity of Dermal Papilla Cells by Suppressing Wnt/β-Catenin Activation.

Dermal papilla (DP) cells play a vital role in hair follicle (HF) development and postnatal hair cycling. However, the abilities are lost on further culture. Recent studies have demonstrated significant influences of posttranscriptional regulation by microRNA (miRNA) on HF development. The current study aims to investigate how miRNAs regulate Wnt/β-catenin to control HF inductivity of DP cells by performing microarray analysis in early- and late-passage DP cells and transfecting with miRNAs inhibitor or mimic. Results showed early-passage DP cells strongly expressed miRNAs related to inhibition of noncanonical Wnt pathways. In late-passage DP cells, miRNAs capable of inhibiting the canonical Wnt/β-catenin pathway were upregulated, in addition to the miRNAs targeting the noncanonical Wnt pathway. Moreover, we verified that β-catenin expression was downregulated by miR-195-5p overexpression in dose manner. Meanwhile LRP6 expression was downregulated in both protein and mRNA as well as the genes involved in the hair inductivity of DP cells. These results suggest that the appearance of miRNAs that suppress the Wnt/β-catenin pathway may be responsible for the loss of ability of DP cells in culture and miR-195-5p is the potential key factor involved in regulating HF inductivity of DP cells.

Comparing the genomes of cutaneous melanoma tumors to commercially available cell lines.

Insulated culture environment and prolonged propagation contribute to known limitations of cell lines, and selection is often limited to availability or favorable growth characteristics. To better characterize and improve selection of cell lines, we compared 60 melanoma cell lines profiled by the Cancer Cell Line Encyclopedia and 472 cutaneous melanoma tumors profiled by The Cancer Genome Atlas by DNA sequence and copy number alterations. All samples were scored for stromal and immune cell composition by the ESTIMATE algorithm, and 412 tumors with ≥ 60% tumor cell fraction were compared to cell lines. Uncharacterized early passage cell lines that lacked BRAF, NRAS, or NF1 mutations had near zero mean Pearson correlation of copy number alterations per gene to tumors and also tended to have higher stromal scores. The Comet Exact Test was applied to tumors and cell lines identifying three pairs of genes mutated in a mutually exclusive pattern in tumors but not cell lines: BRAF and NRAS, BRAF and NF1, as well as NRAS and PTEN. Additionally, 31 genes were more frequently mutated in cell lines than tumors. Avoiding cell lines with co-occurring mutually exclusive mutations and the fewest differentially mutated genes within a known distribution of genetic similarity to tumors by copy number alterations may optimize selection.

Extracellular matrix derived from periodontal ligament cells maintains their stemness and enhances redifferentiation via the wnt pathway.

Large numbers of viable cells cannot be obtained from periodontal ligament tissues of patients with periodontitis. Therefore, it is imperative to establish an ex vivo environment that can support cell proliferation and delay senescence. Here, we have successfully reconstructed a native extracellular matrix (ECM), derived from early-passage human periodontal ligament cells (PDLCs) using the NH4 OH/Triton X-100 protocol. The ECM was investigated by scanning electron microscopy and immunostaining for specific ECM proteins (collagen I and fibronectin). Late-passage ECM-expanded PDLCs exhibited a much higher proliferation index and lower levels of reactive oxygen species (ROS), confirmed by the increased expression of pluripotent markers and enhanced osteogenic capacity. Interestingly, the Wnt pathway was suppressed during the ECM expansion-mediated increase in pluripotency, but was activated in an osteogenic differentiation environment, as confirmed by treatment with the XAV-939 β-catenin inhibitor or the SP600125 c-Jun N-terminal kinase (JNK) inhibitor. Cell sheets formed by ECM-expanded PDLCs exhibited an enhanced periodontal tissue regeneration capacity compared to those formed on tissue culture polystyrene (TCP) surfaces in vivo. Taken together, the cell-free ECM provides a tissue-specific cell niche for the ex vivo expansion of PDLCs while retaining stemness and osteogenic potential, partially via the Wnt pathway. This represents a promising matrix for future applications in periodontal tissue regeneration therapy. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 272-284, 2018.

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