Prolonged Passaging Research Articles

Controlled cell proliferation and immortalization of human dental pulp stem cells with a doxycycline-inducible expression system.

Human dental pulp stem cells are a potentially useful resource for cell-based therapies and tissue repair in dental and medical applications. However, the primary culture of isolated dental pulp stem cells has notably been limited. A major requirement of an ideal human dental pulp stem cell culture system is the preservation of efficient proliferation and innate stemness over prolonged passaging, while also ensuring ease of handling through standard, user-friendly culture methods. In this study, we have engineered a novel human dental pulp stem cell line, distinguished by the constitutive expression of telomerase reverse transcriptase (TERT), and the conditional expression of the R24C mutant cyclin-dependent kinase 4 (CDK4R24C) and Cyclin D1. We have named this cell line Tet-off K4DT hDPSCs. Furthermore, we have conducted a comprehensive comparative analysis of their biological attributes in relation to a previously immortalized human dental pulp stem cells, hDPSC-K4DT, which were immortalized by the constitutive expression of CDK4R24C, Cyclin D1 and TERT. In Tet-off K4DT cells, the expression of the K4D genes can be precisely suppressed by the inclusion of doxycycline. Remarkably, Tet-off K4DT cells demonstrated an extended cellular lifespan, increased proliferative capacity, and enhanced osteogenic differentiation potential when compared to K4DT cells. Moreover, Tet-off K4DT cells had no observable genomic aberrations and also displayed a sustained expression of stem cell markers even at relatively advanced passages. Taken together, the establishment of this new cell line holds immense promise as powerful experimental tool for both fundamental and applied research involving dental pulp stem cells.

Abstract A014: ClpP drives efficacy and mediates acquired resistance with imipridones ONC201 and ONC206 in glioma in vitro

Abstract A014: ClpP drives efficacy and mediates acquired resistance with imipridones ONC201 and ONC206 in glioma <i>in vitro</i>

Unraveling Tumorigenicity in iPSC-Derived Immune Cells: The Impact of Chromosomal Abnormalities and Proliferative Intermediates

Response to tumor cells by the immune system can determine the course of a cancer. This guiding principle and the dynamic, adaptable nature of our immune system underlies the drive to generate immune cells for cancer immunotherapy. Induced pluripotent stem cells (iPSCs) provide an unlimited resource for the generation of immune cells such as T cells and dendritic cells (DCs) for cancer immunotherapy. However, the use of iPSC-derived immune cells in clinical applications has been limited due to concerns of safety. In vitro passage of iPSCs can introduce selective pressures that promote the accumulation of mutations that confer a growth advantage. Over several passages, this can lead to the acquisition of chromosomal abnormalities. Moreover, differentiation protocols of varying efficiency permit the presence of immature cells, contaminating the differentiated cell population. However, the impact of chromosomal abnormalities and intermediate products with proliferation potential on tumorigenicity of iPSC-derived immune cells remains unclear. Here, we investigated the tumorigenic potential of iPSC-derived T cells and DCs in immunocompetent and immunodeficient mice. Pmel-1 T-cell receptor (TCR) transgenic mouse T cells were reprogrammed using Sendai virus vectors carrying Oct3/4, Sox2, Klf4 and cMyc. While our iPSCs were phenotypically normal, even at early passage numbers, various chromosomal abnormalities were found in the resulting cell population. These abnormalities included trisomy 8 and 11 and loss of X chromosome with high frequency. Furthermore, within a population of iPSCs determined to be karyotypically normal by G-banding, unbalanced translocations were found by spectral karyotyping. These findings are consistent with those reported in the literature and highlight the prevalence of chromosomal abnormalities in iPSC populations. As iPSCs can harbor chromosomal abnormalities that are undetectable by standard methods, we assessed the tumorigenic potential of iPSC-derived cells, similar to what may be used in clinical applications. Mouse iPSCs were differentiated into CD8αβ T cells by co-culture with murine OP9 bone marrow stromal cells expressing the Notch ligand Delta-like 1 (OP9-DL1) and were enriched by CD8 positive selection. Activated iPSC-derived T cells were adoptively transferred to immunocompetent C57BL/6 and immunodeficient NOD/SCID, IL-2 receptor γ chain knockout mice. Immunodeficient but not immunocompetent mice developed hepatic tumors. Reverse transcription (RT)-PCR identified Pme1-l TCR gene in tumor cells, confirming tumor origin. Moreover, histology of liver tumors is suggestive of lymphoma or lymphosarcoma. Within the population of CD8+ iPSC-derived cells, a small subpopulation co-expressing CD8 and SSEA-1 was present. Sorting the cells by SSEA-1 positivity eliminated tumor formation in vivo. Mouse iPSCs were also differentiated into DCs by co-culture with OP9-DL1 cells and granulocyte macrophage colony-stimulating factor. Despite similar use of iPSCs with chromosomal abnormalities and prolonged passaging in vitro, SSEA-1+ cells were not identified when they were differentiated to DCs, and iPSC-derived DCs did not form tumors in immunodeficient mice. Taken together, this study revealed the potential risk of tumorigenicity of iPSC-derived immune cells in our preclinical model. The presence of chromosomal abnormalities did not definitively determine risk of tumorigenesis upon transplantation. The relationship between SSEA-1 positivity and tumor formation suggests contaminating cells may carry tumorigenic characteristics. Further work is required to develop differentiation methods that generate pure populations, free of tumorigenic intermediates, to potentiate the clinical use of iPSC-derived immunotherapy.

Minor salivary gland stem cells: a comparative study of the biological properties under clinical-grade culture conditions.

Development of clinical-grade, cell preparations is central to cGMP (good manufacturing practice compliant) conditions. This study aimed to investigate the potential of two serum/xeno-free, cGMP (StemPro, StemMacs) culture media to maintain "stemness" of human minor salivary gland stem cell (mSG-SC) cultures compared to a complete culture medium (CCM). Overall, StemMacs resulted in higher proliferation rates after p.6 compared to the conventional serum-based medium, while StemPro showed substantial delays in cell proliferation after p.9. The mSG-SCs cultures exhibited two distinct cell populations at early passages a mesenchymal subpopulation and an epithelial-like subpopulation. Expression of several markers (CD146, STRO-1, SSEA-4, CD105, CD106, CD34, K 7/8, K14, K18) variably decreased with prolonged passaging (all three media). The percentage of SA-β-gal positive cells was initially higher for StemMacs compared to StemPro/CCM and increased with prolonged passaging in all cases. The telomere fragment length decreased with prolonged passaging in all three media but more pronouncedly for the CCM. Expansion under serum-free conditions caused pronounced upregulation of ALP and BMP-2, with parallel complete elimination of the baseline expressions of LPL (all three media) and ACAN (serum-free media), therefore, showing a preferential shift of the mSG-SCs towards osteogenic phenotypes. Finally, several markers (Nanog, SOX-2, PDX-1, OTX2, GSC, HCG) decreased with prolonged culture, indicating successive loss of "stemness". Based on the findings, it seems that StemPro preserve stemness of the mSG-SCs after prolonged culture. Nevertheless, there is still a vacant role for the ideal development of clinical-grade culture conditions.

Abstract 4914: Role of ClpP in the anti-cancer effects of imipridone ONC201 and ONC206

Abstract ONC201 is the first bitopic dopamine receptor D2 (DRD2) antagonist and allosteric mitochondrial protease ClpP agonist. Downstream of target engagement, ONC201 activates the integrated stress response pathway resulting in selective tumor cell death. ONC201 is well tolerated and induces durable tumor regressions in H3 K27M-mutant glioma patients. ONC206, a derivative of ONC201 currently in Phase I trials for adult and pediatric brain tumors, is also a bitopic DRD2 antagonist and ClpP agonist with nanomolar potency. We further evaluated in vitro efficacy and mechanism of action specific to ClpP for ONC206 and investigated acquired resistance to ONC201 and ONC206. FITC-casein and peptide degradation assays confirmed that ONC206 acts as an agonist of human ClpP with enhanced potency relative to ONC201. Broad efficacy in the nanomolar range for ONC206 (GI50 &amp;lt;78-889nM, 72h) was observed in 1,088 Genomics of Drug Sensitivity in Cancer (GDSC) cancer cell lines with increased sensitivity in cell lines exhibiting high ClpP mRNA expression. Among solid tumors, nervous system-related cell lines were particularly sensitive. Gene Expression Profiling Interactive Analysis (GEPIA) database analysis showed ClpP mRNA was overexpressed in glioblastoma cells relative to normal cells. GDSC observations were confirmed in glioblastoma (GBM) (T98G, A172, U87MG), H3.3 K27M mutant diffuse intrinsic pontine glioma (DIPG) (SF8628, SF7761) and astrocytoma (H4, U118MG) cell lines. ONC206 was more potent than ONC201 in cell viability assays for all glioma lines tested. SF8628 cells with a CRISPR-mediated ClpP knockout were shown to demonstrate resistance to both ONC201 and ONC206. Polyclonal resistant cell lines were generated through prolonged passaging in increasing concentrations of ONC201 or ONC206 (SF8628 and T98G cells). Cells with acquired resistance to ONC201 (ONC201Res) or ONC206 (ONC206Res) showed cross resistance to both imipridones. Time-stability of resistance was confirmed by removing drug for 2 to 4 weeks and retesting. RNAseq analysis revealed upregulation of the integrated stress response with imipridone treatment in T98G parental cells but not resistant clones. Whole genome sequencing revealed ClpP mutations in both ONC201Res and ONC206Res cells. Our results confirm the role of ClpP in the mechanism of action of ONC201 and ONC206 in tumor cells. Citation Format: Andrew Lee, Cristina Maranto, Scott Foster, Sara Morrow, Joshua E. Allen, Randall Lanier, Phiroze Sethna, Varun V. Prabhu. Role of ClpP in the anti-cancer effects of imipridone ONC201 and ONC206. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4914.

Astroglial differentiation of fibronectin‑positive human "glia-like" cells.

Fibronectin (Fn) is a glycoprotein of extracellular matrix produced by a variety of mesenchymal and neoplastic cell types. In adult brain tissue, Fn is restricted to blood vessels. However, adult human brain cultures are almost entirely comprised of flat or spindle‑shaped Fn-positive cells usually referred to as "glia-like" cells. Since Fn is primarily present in fibroblasts, these cultures may be considered to be of non-glial origin. Cells gained by long-term culturing of adult human brain tissue derived from brain biopsies obtained from 12 patients with non-malignant diagnoses were examined by immunofluorescence methods. Primary cultures contained GFAP-/Vim+/Fn+ "glia-like" cells (95-98 %) and GFAP+/Vim+/Fn- astrocytes (0.1 %) which disappeared by passage number 3. The formation of cell processes and enlargement of cell bodies was observed in 9 of 12 cultures with decreased cell growth during passages 12 to 17. It is remarkable that during this period, all "glia-like" cells became GFAP+/Vim+/Fn+. Herein, we confirm our previously published hypothesis about the origin of adult human "glia-like" cells, which we consider to be precursor cells scattered through the brain cortex and subcortical white matter. Cultures were comprised entirely of GFAP-/Fn+ "glia-like" cells and showed morphological and immunochemical astroglial differentiation with spontaneously decelerated growth during prolonged passaging. We propose that the adult human brain tissue contains a "dormant population" of undefined glial precursor cells. Under culture, these cells show to have a high proliferative capacity and different stages of cell dedifferentiation (Fig. 2, Ref. 21).

Defective RNA Particles of Plant Viruses-Origin, Structure and Role in Pathogenesis.

The genomes of RNA viruses may be monopartite or multipartite, and sub-genomic particles such as defective RNAs (D RNAs) or satellite RNAs (satRNAs) can be associated with some of them. D RNAs are small, deletion mutants of a virus that have lost essential functions for independent replication, encapsidation and/or movement. D RNAs are common elements associated with human and animal viruses, and they have been described for numerous plant viruses so far. Over 30 years of studies on D RNAs allow for some general conclusions to be drawn. First, the essential condition for D RNA formation is prolonged passaging of the virus at a high cellular multiplicity of infection (MOI) in one host. Second, recombination plays crucial roles in D RNA formation. Moreover, during virus propagation, D RNAs evolve, and the composition of the particle depends on, e.g., host plant, virus isolate or number of passages. Defective RNAs are often engaged in transient interactions with full-length viruses-they can modulate accumulation, infection dynamics and virulence, and are widely used, i.e., as a tool for research on cis-acting elements crucial for viral replication. Nevertheless, many questions regarding the generation and role of D RNAs in pathogenesis remain open. In this review, we summarise the knowledge about D RNAs of plant viruses obtained so far.

EXTH-104. ROLE OF CLPP AND MITOCHONDRIAL METABOLISM IN THE ANTI-CANCER EFFECTS OF IMIPRIDONE ONC201 AND ONC206 IN GLIOBLASTOMA AND DIPG

Abstract ONC201 is the first bitopic dopamine receptor D2 (DRD2) antagonist and allosteric mitochondrial protease ClpP agonist. ONC201 is well tolerated and induces durable tumor regressions in H3 K27M-mutant glioma patients. ONC206, a derivative of ONC201 currently in Phase I trials, is also a bitopic DRD2 antagonist and ClpP agonist with enhanced nanomolar potency relative to ONC201. We evaluated in vitro efficacy, mechanism(s) of action and acquired resistance for ONC201 and ONC206 in glioblastoma (GBM) (T98G, A172, U87MG), H3.3 K27M mutant DIPG (SF8628, SF7761) and astrocytoma (H4, U118MG) cell lines. ONC206 was more potent than ONC201 in cell viability assays for all line. A mutation in ClpP has previously been associated with resistance to imipridones. Accordingly, SF8628 cells with a CRISPR-mediated ClpP knockout were resistant to both ONC201 and ONC206. Polyclonal resistant cell lines were generated through prolonged passaging in increasing concentrations of ONC201 (SF8628 and T98G cells) or ONC206 (T98G cells). Cells with acquired resistance to ONC201 (ONC201Res) or ONC206 (ONC206Res) showed cross resistance to both imipridones. Time-stability of resistance was confirmed by removing drug for 2 to 4 weeks and retesting. Whole genome sequencing revealed ClpP mutations in both ONC201Res and ONC206Res T98G cells. RNAseq analysis revealed upregulation of the integrated stress response, apoptosis pathways, and amino acid metabolism with imipridone treatment in T98G parental cells but not resistant clones. Depleting glucose in the media enhanced cell viability inhibition by imipridones, indicating mitochondrial metabolism may contribute to their mechanism of action. Our results identify ClpP and mitochondrial metabolism as key aspects for the mechanism of action of ONC201 and ON206 in DIPG and GBM cell lines.

Enzyme-free targeted DNA demethylation using CRISPR-dCas9-based steric hindrance to identify DNA methylation marks causal to altered gene expression.

DNA methylation involves the enzymatic addition of a methyl group primarily to cytosine residues in DNA. This protocol describes how to produce complete and minimally confounded DNA demethylation of specific sites in the genome of cultured cells by clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 and without the involvement of an epigenetic-modifying enzyme, the purpose of which is the evaluation of the functional (i.e., gene expression or phenotypic) consequences of DNA demethylation of specific sites that have been previously implicated in particular pathological or physiological contexts. This protocol maximizes the ability of the easily reprogrammable CRISPR-dCas9 system to assess the impact of DNA methylation from a causal rather than correlational perspective: alternative protocols for CRISPR-dCas9-based site-specific DNA methylation or demethylation rely on the recruitment of epigenetic enzymes that exhibit additional nonspecific activities at both the targeted site and throughout the genome, confounding conclusions of causality of DNA methylation. Inhibition or loss of DNA methylation is accomplished by three consecutive lentiviral transductions. The first two lentiviruses establish stable expression of dCas9 and a guide RNA, which will physically obstruct either maintenance or de novo DNA methyltransferase activity at the guide RNA target site. A third lentivirus introduces Cre recombinase to delete the dCas9 transgene, which leads to loss of dCas9 from the target site, allowing transcription factors and/or the transcription machinery to interact with the demethylated target site. This protocol requires 3-8 months to complete owing to prolonged cell passaging times, but there is little hands-on time, and no specific skills beyond basic molecular biology techniques are necessary.

Colonoids From Patients With Pediatric Inflammatory Bowel Disease Exhibit Decreased Growth Associated With Inflammation Severity and Durable Upregulation of Antigen Presentation Genes.

Defining epithelial cell contributions to inflammatory bowel disease (IBD) is essential for the development of much needed therapies for barrier repair. Children with very early onset (VEO)-IBD have more extensive, severe, and refractory disease than older children and adults with IBD and, in some cases, have defective barrier function. We therefore evaluated functional and transcriptomic differences between pediatric IBD (VEO and older onset) and non-IBD epithelium using 3-dimensional, biopsy-derived organoids. We measured growth efficiency relative to histopathological and clinical parameters in patient enteroid (ileum) and colonoid (colon) lines. We performed RNA-sequencing on patient colonoids and subsequent flow cytometry after multiple passages to evaluate changes that persisted in culture. Enteroids and colonoids from pediatric patients with IBD exhibited decreased growth associated with histological inflammation compared with non-IBD controls. We observed increased LYZ expression in colonoids from pediatric IBD patients, which has been reported previously in adult patients with IBD. We also observed upregulation of antigen presentation genes HLA-DRB1 and HLA-DRA, which persisted after prolonged passaging in patients with pediatric IBD. We present the first functional evaluation of enteroids and colonoids from patients with VEO-IBD and older onset pediatric IBD, a subset of which exhibits poor growth. Enhanced, persistent epithelial antigen presentation gene expression in patient colonoids supports the notion that epithelial cell-intrinsic differences may contribute to IBD pathogenesis.

Nrf2 dependent antiaging effect of milk-derived bioactive peptide in old fibroblasts.

Prolonged passaging of primary fibroblast cells totally shapes the natural biological phenomena and leads to the appearance of features related to senescence. As a result, it is a good natural tool to delineate the molecular mechanism of cellular aging. The present investigation revealed the antiaging effect of milk-derived novel bioactive peptide (VLPVPQK). The peptide played an important role in downregulating apoptosis-related markers in late passages of cultured fibroblast cells. The peptide treatment to aged fibroblasts caused enhancement in cell migration, DNA integrity, and decrease in the lipid peroxidation, reactive oxygen species, nitric oxide production as well as pro-inflammatory cytokines, TNF-α and IL-6. Moreover, the peptide decreased the expression of apoptotic caspases, Bax, and senescence-associated β-galactosidase (SA-β-gal) proteins. The peptide pretreatment also enhanced the extracellular collagen protein and antiapoptotic, Bcl-xL. In addition, the peptide treatment reversed the senescence-related activity in fibroblasts by stimulating Nrf2 mediated antioxidative defense system and inhibiting the action of NFkB/p38MAPK signaling, similar to the commercially available inhibitor (SB203580) of p38MAPK. Thus, the peptide exhibits the antiaging effect in dermal fibroblast cells.

Morphological adaptations in breast cancer cells as a function of prolonged passaging on compliant substrates.

Standard tissue culture practices involve propagating cells on tissue culture polystyrene (TCP) dishes, which are flat, 2-dimensional (2D) and orders of magnitude stiffer than most tissues in the body. Such simplified conditions lead to phenotypical cell changes and altered cell behaviors. Hence, much research has been focused on developing novel biomaterials and culture conditions that more closely emulate in vivo cell microenvironments. In particular, biomaterial stiffness has emerged as a key property that greatly affects cell behaviors such as adhesion, morphology, proliferation and motility among others. Here we ask whether cells that have been conditioned to TCP, would still show significant dependence on substrate stiffness if they are first pre-adapted to a more physiologically relevant environment. We used two commonly utilized breast cancer cell lines, namely MDA-MB-231 and MCF-7, and examined the effect of prolonged cell culturing on polyacrylamide substrates of varying compliance. We followed changes in cell adhesion, proliferation, shape factor, spreading area and spreading rate. After pre-adaptation, we noted diminished differences in cell behaviors when comparing between soft (1 kPa) and stiff (103 kPa) gels as well as rigid TCP control. Prolonged culturing of cells on complaint substrates further influenced responses of pre-adapted cells when transferred back to TCP. Our results have implications for the study of stiffness-dependent cell behaviors and indicate that cell pre-adaptation to the substrate needs consideration.

Establishing estrogen-responsive mouse mammary organoids from single Lgr5+ cells

Recent evidence suggests that mammary cells expressing R-spondin receptor and Wnt pathway regulator Lgr5, regarded as a stem cell marker in multiple tissues, might represent mammary stem cells (MaSCs). Whether L gr5 marks a multipotent subpopulation of Lin-CD24low/medCD49fhigh MaSCs remains controversial. To some extent the differing results reflect different assays used to assess properties of stemness, including lineage tracing in vivo, mammosphere culture, and mammary fat pad transplantation assays. To address this issue directly, we isolated Lgr5+ cells from mammary glands of Lgr5-lacZ mice and established organoids based on principles adapted from studies of Wnt-driven Lgr5+ cell populations in other organs. Mammary organoids were grown from single Lgr5+ mammary cells in Matrigel, the substratum of choice for intestinal organoids, and in a growth factor cocktail containing EGF, Wnt3a and R-spondin, designed to optimally activate the endogenous Wnt signaling program of stem cells. Colonies derived from single Lgr5+ cells manifest at least four distinct cell populations: Lgr5+ and Lgr5- basal cells and c-Kit+ and c-Kit- luminal cells that spontaneously organize into a ductal structure with basal cells around the periphery and luminal cells lining an interior cavity, reminiscent of normal mammary duct structure. Lgr5+ cell-derived organoids were sustainable during prolonged passaging. In contrast, although Lgr5- cells expand into primary colonies, colony-forming efficiency immediately dissipated upon passaging. Furthermore, reproductive hormones induce epithelial cell proliferation resulting in marked increases in lumen diameter accompanied by squamous transdifferentiation. We propose this estrogen-responsive, self-organizing duct-like structure derived from single murine Lgr5+ mammary cells represents a “mini-breast” organoid.

Apoptotic transition of senescent cells accompanied with mitochondrial hyper-function.

Defined as stable cell-cycle arrest, cellular senescence plays an important role in diverse biological processes including tumorigenesis, organismal aging, and embryonic development. Although increasing evidence has documented the metabolic changes in senescent cells, mitochondrial function and its potential contribution to the fate of senescent cells remain largely unknown. Here, using two in vitro models of cellular senescence induced by doxorubicin treatment and prolonged passaging of neonatal human foreskin fibroblasts, we report that senescent cells exhibited high ROS level and augmented glucose metabolic rate concomitant with both morphological and quantitative changes of mitochondria. Furthermore, mitochondrial membrane potential depolarized at late stage of senescent cells which eventually led to apoptosis. Our study reveals that mitochondrial hyper-function contributes to the implementation of cellular senescence and we propose a model in which the mitochondrion acts as the key player in promoting fate-determination in senescent cells.

Influence of Different Components of the Tumor Microenvironment on Human Patient-Derived Lymphoma Cell Engraftment in Immmunodeficient Mice

Patient-derived xenografts (PDX) have become reliable tools for preclinical research for many types of cancer. We recently reported the establishment of 4 human diffuse-large B-cell lymphoma (DLBCL) PDX in mice arising from patient prostate carcinoma tissue (Wetterauer,C. et al. 2015). All PDX were EBV associated and of ABC subtype as determined by the new algorithm of Choi et al. 2009. In contrast, none of the donor patients showed clinical or histological evidence of lymphoma disease. To address the question why lymphoma engraftment takes place in immunodeficient mice whereas patients do not show clinical evidence of disease, we compared growth behavior of the DLBCL PDX in 4 different mouse strains harboring different immunological deficiencies. Furthermore using 3 different implantation routes the dissemination behavior of these models into different immunological niches was evaluated. Results were compared to a similar study carried out with an EBV negative DLBCL PDX of a secondary, cerebral lymphoma (ABC-subtype).None of 4 EBV+ DLBCL PDX showed tumor growth after implantation in NMRI nu/nu (B- cells+, T cells-, NK cells+, n=5 mice/PDX) or 1147-F mice (B cells-, T cells+, NK cells+ n=5 mice/PDX). Subcutaneous implantation into NOD/SCID mice (B cells-, T cells-, NK cells+ n=5 mice/PDX) revealed tumor growth for 2 of 4 lymphomas displaying take rates of 80% (4 out of 5 mice) and 100% (5 out of 5 mice) within one model. Infiltration of murine NK cells in tumor tissue was determined by IHC (analyses ongoing). All 4 EBV+ lymphoma models showed stable growth in NOG mice (B cells-, T cells-, NK cells- n= 10 - 12 mice/PDX) with a take rate of 100% and an average passaging time of 20 days, ranging from 14 to 26 depending on the specific model. Taken together these results indicate that tumor growth of EBV+ lymphomas in mice depends on the NK cell activity as well as the B- and T cells status of the recipient mouse. These cell populations control tumor engraftment in mice, which might explain the absence of lymphoma disease in donor patients. In contrast the PDX of a clinically apparent EBV- DLBCL patient was able to circumvent NK cell block in NMRI nu/nu mice and showed similar take rates (100%, 5 out of 5 engrafted mice) but prolonged passaging times (35 days) as compared to NOG (26 days).Comparison of tumor cell engraftment after subcutaneous (s.c.), intravenous (i.v.) and intratibial (i.t.) injection of tumor cells in NOG mice revealed disseminated tumor growth for the EBV- DLBCL PDX exclusively when injected i.v. or i.t.. After subcutaneous implantation of lymphoma material, no tumor cells could be detected by flow cytometry or IHC in lymph nodes, blood, spleen, brain, liver, lung or bone marrow of recipient mice. However, lymphoma cells could be detected in liver and bone marrow of all (n= 8) i.v. and i.t. injected mice. Currently we are analyzing the dissemination behavior of the EBV+ lymphomas after injection into the different immunological niches as described above.In summary these data highlight the importance of the tumor microenvironment in lymphoma engraftment and dissemination in vivo. Accordingly we could show for our EBV associated lymphoma models that NK-, T- and B cell population control engraftment in vivo and are possibly involved in controlling EBV+ transformed lymphatic cells in patients. Furthermore, we showed that injection into the biologically relevant niche enables lymphoma cells to form multiple tumor nodules in distinct organs in vivo. These models contribute to a better understanding of the interplay between lymphoma cells and their microenvironment. They will thereby facilitate the discovery of new targets for innovative anti-lymphoma treatment strategies. DisclosuresTschuch:Oncotest GmbH: Employment. Klingner:Oncotest GmbH: Employment. Lenhard:Oncotest GmbH: Employment. Haapaniemi:Biositehisto Oy: Employment. Schueler:Oncotest GmbH: Employment.

Deep sequencing analysis of the heterogeneity of seed and commercial lots of the bacillus Calmette-Guérin (BCG) tuberculosis vaccine substrain Tokyo-172.

BCG, only vaccine available to prevent tuberculosis, was established in the early 20th century by prolonged passaging of a virulent clinical strain of Mycobacterium bovis. BCG Tokyo-172, originally distributed within Japan in 1924, is one of the currently used reference substrains for the vaccine. Recently, this substrain was reported to contain two spontaneously arising, heterogeneous subpopulations (Types I and II). The proportions of the subpopulations changed over time in both distributed seed lots and commercial lots. To maintain the homogeneity of live vaccines, such variations and subpopulational mutations in lots should be restrained and monitored. We incorporated deep sequencing techniques to validate such heterogeneity in lots of the BCG Tokyo-172 substrain without cloning. By bioinformatics analysis, we not only detected the two subpopulations but also detected two intrinsic variations within these populations. The intrinsic variants could be isolated from respective lots as colonies cultured on plate media, suggesting analyses incorporating deep sequencing techniques are powerful, valid tools to detect mutations in live bacterial vaccine lots. Our data showed that spontaneous mutations in BCG vaccines could be easily monitored by deep sequencing without direct isolation of variants, revealing the complex heterogeneity of BCG Tokyo-172 and its daughter lots currently in use.

Host Adaptation and the Alteration of Viral Properties of the First Influenza A/H1N1pdm09 Virus Isolated in Japan.

A/Narita/1/2009 (A/N) was the first H1N1 virus from the 2009 pandemic (H1pdm) to be isolated in Japan. To better understand and predict the possible development of this virus strain, the effect of passaging A/N was investigated in Madin-Darby canine kidney cells, chicken eggs and mice. A/N that had been continuously passaged in cells, eggs, or mice obtained the ability to grow efficiently in each host. Moreover, A/N grown in mice had both a high level of pathogenicity in mice and an increased growth rate in cells and eggs. Changes in growth and pathogenicity were accompanied by amino acid substitutions in viral hemagglutinin (HA) and PB2. In addition, the adapted viruses exhibited a reduced ability to react with ferret antisera against A/N. In conclusion, prolonged passaging allowed influenza A/N to adapt to different hosts, as indicated by a high increase in proliferative capacity that was accompanied by an antigenic alteration leading to amino acid substitutions.

Phenotypic and Functional Properties of Porcine Dedifferentiated Fat Cells during the Long-Term Culture In Vitro.

It has been proved that terminally differentiated mature adipocytes possess abilities to dedifferentiate into fibroblast-like progeny cells with self-renewal and multiple differentiation, termed dedifferentiated fat (DFAT) cells. However, the biological properties of DFAT cells during long-term culture in vitro have not been elucidated. Here, we obtained fibroblast-like morphology of porcine DFAT cells by ceiling culture. During the dedifferentiation process, round mature adipocytes with single large lipid droplets changed into spindle-shaped cells accompanied by the adipogenic markers PPARγ, aP2, LPL, and Adiponectin significant downregulation. Flow cytometric analysis showed that porcine DFAT cells displayed similar cell-surface antigen profile to mesenchymal stem cells (MSCs). Furthermore, different passages of porcine DFAT cells during long-term culture in vitro retained high levels of cell viabilities (>97%), efficient proliferative capacity including population doubling time ranged from 20 h to 22 h and population doubling reached 47.40 ± 1.64 by 58 days of culture. In addition, porcine DFAT cells maintained the multiple differentiation capabilities into adipocytes, osteoblasts, and skeletal myocytes and displayed normal chromosomal karyotypes for prolonged passaging. Therefore, porcine DFAT cells may be a novel model of stem cells for studying the functions of gene in the different biological events.

X chromosome inactivation in human parthenogenetic embryonic stem cells following prolonged passaging.

The present study aimed to investigate the X chrochromosome inactivation (XCI) status in long-term cultured human parthenogenetic embryonic stem cells. One human embryonic stem (hES) cell line and 2 human parthenogenetic embryonic stem (hPES) cell lines were subjected to long-term culture in vitro (>50 passages). Karyotyping, array-based comparative genomic hybridization (aCGH), X-inactive specific transcript (XIST) RNA, immunofluorescence staining and real-time PCR were used to assess the chromosome karyotypes of these cells and the XCI status. X chromosome microdeletion was observed in the hPES-2 cells following culture for 50 passages. As early as 20 passages, XIST RNA expression was detected in the hPES-2 cells and was followed by low X-linked gene expression. The XIST RNA expression level was higher in the differentiated hPES-2 cells. The hPES-2′ cells that were subclones of hPES-2 retained the XCI status, and had low XIST and X-linked gene expression. XIST RNA expression remained at a low level in the differentiated hPES-2′ cells. The human biparental embryonic stem (hBES)-1 and hPES-1 cells did not exhibit XCI, and the differentiated hPES-1 cells had high expression levels of XIST RNA. In conclusion, the chromosome karyotypes of some hPES cell lines revealed instabilities. Similar to the hES cells, the hPES cells exhibited 3 XCI statuses. The unstable XCI status of the hPES-2 line may have been related to chromosome instability. These unstable chromosomes renedered these cells susceptible to environmental conditions and freezing processes, which may be the result of environmental adaptations.

TGF-β1 induces differentiation of papillary fibroblasts to reticular fibroblasts in monolayer culture but not in human skin equivalents

Fibroblasts isolated from the papillary and reticular dermis are different from each other in vitro. If papillary fibroblasts are subjected to prolonged serial passaging they will differentiate into reticular fibroblasts. Reticular fibroblasts have been shown to resemble myofibroblasts in several ways. TGF-β1 is the most important factor involved in myofibroblast differentiation. we investigated if TGF-β1 can induce differentiation of papillary fibroblasts into reticular fibroblasts, in monolayer cultures and in human skin equivalents. Monolayer cultures of and human skin equivalents generated with papillary fibroblasts were stimulated with TGF-β1. The expression of markers specific for reticular and papillary fibroblasts was measured by qPCR and immunohistochemical analysis in monolayer cultures. In human skin equivalents, the morphology and the expression of several markers was analysed and compared to untreated papillary and reticular human skin equivalents. Monolayer cultures of papillary fibroblasts started to express a reticular marker profile after stimulation with TGF-β1. Human skin equivalents generated with papillary fibroblast and stimulated with TGF-β1 were similar to papillary control equivalents and did not obtain reticular characteristics. Expression of reticular markers was only found in the lower layers of TGF-β1-stimulated papillary skin equivalents. TGF-β1 can induce differentiation to reticular fibroblasts in monolayer cultures of papillary fibroblasts. In skin equivalents no such effects were found. The major difference between these experiments is the presence of extracellular matrix in skin equivalents. Therefore, we hypothesize that the matrix secreted by papillary fibroblasts protects them from TGF-β1 induced differentiation.