Reprogramming of somatic common marmoset (Callithrix jacchus) cells towards pluripotency

Da iPS Zellen in jeden Zell- oder Gewebetyp differenziert werden können, der für eine autologe Transplantation benötigt wird, wären induzierte pluripotente Stammzellen in der regenerativen Medizin von großem Nutzen. Daher ist es sinnvoll, zur Erforschung der Generierung (und späteren Differenzierung) von iPS Zellen Tiermodelle einzusetzen, die näher mit dem Menschen verwandt sind als das üblicherweise verwendete Mausmodell. Deshalb war es Ziel der vorliegenden Arbeit, adulte Hautfibroblasten des Weißbüschelaffen (Callithrix jacchus) in iPS Zellen zu überführen. Da zu Beginn der Forschungsarbeiten keine Veröffentlichungen zur Reprogrammierung von Callithrix jacchus Zellen vorlagen, wurden die verwendeten Methoden aus etablierten Praktiken in der Forschung an Menschen- und Mauszellen abgeleitet und dem Affenmodell angepasst. Dabei wurden unterschiedliche lentivirale Methoden und Reprogrammierungs-bedingungen getestet. Die Zellen wurden einerseits mit einem polycistronischen Vek-tor, der die cDNA der humanen Yamanaka-Faktoren OCT4, KLF4, SOX2 und MYC koexprimierte, transduziert. Andererseits wurde der Reprogrammierungsprozess mit separaten Vektoren initiiert, mit Hilfe derer die genannten Faktoren einzeln in die Zelle eingebracht wurden. Weitere Ansätze wurden unter Hinzunahme eines LIN28A-Expressionsvektors durchgeführt. Zur Unterstützung des Reprogrammierungspro-zesses wurden sogenannte Small Molecules zu den Zellen gegeben, die epigenetische Blockaden beseitigen oder Signaltransduktionswege aktivieren oder inhibieren. Allerdings konnten nur partiell reprogrammierte Zellen (piPS) generiert werden, die auch durch die Verwendung von Small Molecules nicht in vollständige iPS Zellen über-führt werden konnten. Die generierten Kolonien zeigten allerdings Charakteristika pluripotenter Zellen. So exprimierten sie die Alkalische Phosphatase und wurden positiv auf SSEA3- und SSEA4-exprimierende Zellen getestet. Dabei exprimierten aber nur wenige Zellen die Marker TRA-1-81 oder TRA-1-60. Die Analyse der endogenen pluripotenzassoziierten Transkripte in den piPS Zellen mittels qRT-PCR zeigte hingegen, dass diese Gene auf ein höheres Level als in den Fibroblasten induziert wurden. Allerdings wurde je nach Methode nur eine mit ES Zellen vergleichbare Expression von SOX2, MYC und ZFP24 oder KLF4 gemessen; die Hochregulation von POU5F1 war dabei stets unzureichend. Zudem wurde der retrovirale Vektor in den piPS Zellen nicht stillgelegt. Die Hauptkomponentenanalyse (PCA), die auf einer globalen Transkriptomanalyse aufbaut, zeigte, dass die piPS Zellen eine miteinander vergleichbare Genexpression aufwiesen. Im Vergleich zu ES Zellen zeigten sie zwar deutliche Unterschiede, ihre Transkriptome waren diesen aber ähnlicher als denen der Hautfibroblasten. Auch wenn die somatischen Weißbüschelaffenzellen nicht vollständig reprogrammiert werden konnten, können die generierten piPS Zellen für die Identifizierung transkriptioneller und epigenetischer Veränderung sowie aktiver und inhibierter Signalt-ransduktionswege während der unterschiedlichen Stadien des Reprogrammierungsprozesses eingesetzt werden. Aufbauend auf den Ergebnissen dieser Arbeit könnte in weiteren Forschungen versucht werden, Keratinozyten mit sechs Faktoren auf separaten Vektoren zu transduzieren. Die epithelialen Zellen könnten möglicherweise effizienter reprogrammiert werden, da sie die MET nicht durchlaufen. Auch wenn der polycistronische Vektor zu besseren Reprogrammierungsergebnissen führte, können mit separaten Vektoren mehr als vier Faktoren in die Zellen transduziert werden.Induced pluripotent stem cells (iPSCs) are attractive for regenerative medicine because they can be differentiated towards any somatic cell type and tissues needed for autolo-gous transplantation. It would be desirable to test the medical applicability of this notion in a relevant animal model system which is more closely related to humans than the commonly used mouse model is. Thus the non-human primate common marmoset (Callithrix jacchus) is a suitable choice. The major aim of this thesis was to reprogram dermal fibroblasts of the marmoset monkey towards iPSCs. Because the conditions for reprogramming marmoset cells were illdefined at the beginning of this research project, different lentiviral approaches and reprogramming conditions were used. Thus the somatic cells were transduced with a polycistronic vector, co-expressing the cDNAs for human OCT4, KLF4, SOX2 and MYC (Yamanaka factors). Furthermore the reprogramming process was initiated with separate vectors, each coding a Yamanaka factor together with a different fluorescence protein. In further approaches these were additionally combined with a LIN28A-expression vector. To overcome possible epigenetic roadblocks due to remaining DNA-methylation or to activate or inhibit signaling trans-duction pathways, which are supportive for pluripotency, small molecules were added to the cells. However, the marmoset skin fibroblasts could not be fully reprogrammed with all the different approaches. Even the use of various small molecules did not yield to fully reprogrammed cells. Indeed the obtained colonies showed some hallmarks of pluripotency. They expressed alkaline phosphatase and stained positive for the pluripotency markers SSEA3 and SSEA4, but they were mostly negative for TRA-1-81 and TRA-1-60 expression. Analysis of endogenous pluripotency associated gene transcription by qRT-PCR showed that these genes were significantly induced above levels of unmanipulated fibroblasts. However, depending on the approach, only SOX2, MYC and ZFP24 or KLF4 expression was comparable between the partial iPSCs (piPSCs) and marmoset embryonic stem cells (ESC). Beyond that especially the up regulation of POU5F1 expression was insufficient. Noteworthy, they also failed to silence retroviral vector expression, a phenomenon normally observed during successful pluripotency induction in human and mouse cells. Furthermore, global transcriptome measurements by RNA-seq and subsequent principle component analysis (PCA) revealed that the generated piPS cells showed comparable gene expression among each other but still different from marmoset ESCs. However, they were much more similar to the ESCs than to the skin fibroblasts indicating their partially reprogrammed state. Thus piPSCs are not fully reprogrammed they are very powerful to identify transcrip-tional and epigenetic changes as well as active and inhibited signal transduction path-ways during the different stages of the reprogramming process. However, the findings of the piPSC analyses will certainly help to find better ways for an efficient and complete reprogramming of somatic marmoset cells. One could consider transducing kerationcytes with six factors on six separate vectors. The epithelial cells could possibly be reprogrammed more efficiently, because they do not have to pass the MET. Alt-hough the polycistronic vector led to better results, only separate vectors are suitable to transduce cells with more than four factors

Differential profile of ultrasound findings associated with malignancy in mixed and solid thyroid nodules in an elderly female population

Objective. Ultrasonographic characteristics are associated with thyroid malignancy. Our aim was to compare the diagnostic value of ultrasound features in the detection of thyroid malignancy in both solid and mixed nodules. Methods. We prospectively studied female patients (≥50 years) referred to ultrasound-guided fine needle aspiration biopsy. Ultrasound features considered suspicious were hypoechogenicity, microcalcifications, irregular margins, high anteroposterior (AP)/axial-ratio, and absent halo. Associations were separately assessed in mixed and solid nodules. Results. In a group of 504 elderly female patients (age =  years), the frequency of malignant cytology was 6%. Thirty-one percent of nodules were mixed and 60% were solid. The rate of malignant cytology was similar for mixed and solid nodules (7.4 versus 5.8%, : 0.56). While in mixed nodules none of the ultrasound characteristics were associated with malignant cytology, in solid nodules irregular margins and microcalcifications were significant (all ). The combination of irregular margins and/or microcalcifications significantly increased the association with malignant cytology only in solid nodules (OR: 2.76 (95% CI: 1.25–6.10), : 0.012). Conclusions. Ultrasound features were of poor diagnostic value in mixed nodules, which harbored malignant lesions as often as solid nodules. Our findings challenge the recommended minimal size for ultrasound-guided fine needle aspiration biopsy in mixed nodules.Fil: Vera, María Inés. Dr. Cesar Milstein Hospital; ArgentinaFil: Meroño, Tomás. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Urrutia, María Agustina. Dr. Cesar Milstein Hospital; ArgentinaFil: Parisi, Carina. Dr. Cesar Milstein Hospital; ArgentinaFil: Morosan, Yanina. Dr. Cesar Milstein Hospital; ArgentinaFil: Rosmarin, Melanie. Dr. Cesar Milstein Hospital; ArgentinaFil: Schnitman, Marta. Dr. Cesar Milstein Hospital; ArgentinaFil: Brites, Fernando Daniel. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Grisendi, Silvio. Dr. Cesar Milstein Hospital; ArgentinaFil: Serrano, María Sol. Dr. Cesar Milstein Hospital; ArgentinaFil: Luciani, Wilfredo. Dr. Cesar Milstein Hospital; ArgentinaFil: Serrano, Leonardo. Dr. Cesar Milstein Hospital; ArgentinaFil: Zuk, Carlos. Dr. Cesar Milstein Hospital; ArgentinaFil: de Barrio, Guillermo. Dr. Cesar Milstein Hospital; ArgentinaFil: Cejas, Claudia. Dr. Cesar Milstein Hospital; ArgentinaFil: Faingold, María Cristina. Dr. Cesar Milstein Hospital; ArgentinaFil: Brenta, Gabriela. Dr. Cesar Milstein Hospital; Argentin

In Vitro Generation of Vascular Wall-Resident Multipotent Stem Cells of Mesenchymal Nature from Murine Induced Pluripotent Stem Cells

Summary: The vascular wall (VW) serves as a niche for mesenchymal stem cells (MSCs). In general, tissue-specific stem cells differentiate mainly to the tissue type from which they derive, indicating that there is a certain code or priming within the cells as determined by the tissue of origin. Here we report the in vitro generation of VW-typical MSCs from induced pluripotent stem cells (iPSCs), based on a VW-MSC-specific gene code. Using a lentiviral vector expressing the so-called Yamanaka factors, we reprogrammed tail dermal fibroblasts from transgenic mice containing the GFP gene integrated into the Nestin-locus (NEST-iPSCs) to facilitate lineage tracing after subsequent MSC differentiation. A lentiviral vector expressing a small set of recently identified human VW-MSC-specific HOX genes then induced MSC differentiation. This direct programming approach successfully mediated the generation of VW-typical MSCs with classical MSC characteristics, both in vitro and in vivo. : In this article, Klein and colleagues show that iPSCs generated from skin fibroblasts of transgenic mice carrying a GFP gene under the control of the endogenous Nestin promoter to facilitate lineage tracing (NEST-iPSCs) can be directly programmed toward mouse vascular wall-typical multipotent mesenchymal stem cells (VW-MSC) by ectopic lentiviral expression of a previously defined VW-MSC-specific HOX code. Keywords: vascular wall-derived mesenchymal stem cells, HOX gene, induced pluripotent stem cells, direct programming, nesti

Exploring sub-lethal effects of exposure to a nucleopolyhedrovirus in the speckled wood (Pararge aegeria) butterfly

This study investigated the sub-lethal effects of larval exposure to baculovirus on host life history and wing morphological traits using a model system, the speckled wood butterfly Pararge aegeria (L.) and the virus Autographa californica nucleopolyhedrovirus. Males and females showed similar responses to the viral infection. Infection significantly reduced larval growth rate, whilst an increase in development time allowed the critical mass for pupation to be attained. There was no direct effect of viral infection on the wing morphological traits examined. There was, however, an indirect effect of resisting infection; larvae that took longer to develop had reduced resource investment in adult flight muscle mass

Comparison of different systems of ultrasound (US) risk stratification for malignancy in elderly patients with thyroid nodules. Real world experience

To comparatively assess the performance of three sonographic classification systems, American Thyroid Association (ATA), the American College of Radiology Thyroid Imaging Reporting and Data System (ACR TI-RADS), and American Association of Clinical Endocrinologists (AACE)/American College of Endocrinology (ACE)/Associazione Medici Endocrinologi (AME) in identifying malignant nodules in an elderly population. Methods: Cross-sectional study of patients referred for fine needle aspiration biopsy in an academic center for the elderly. One nodule/patient was considered. Nodules classified Bethesda V/VI were considered malignant. Receiver operating characteristics (ROC) curves were established and compared to evaluate diagnostic performance. Malignancy among biopsies below the size cutoff for each ultrasound classification was also compared. Results: One thousand, eight hundred sixty-seven patients (92% females); median (Q1-Q3), age 71 (67-76) years, were studied showing 82.8% benign (Bethesda II) and 2.6% malignant cytology. The three classifications correctly identified malignancy (P < 0.01). Nonetheless, in the ATA and AACE/ACE/AME 16 and 2 malignant nodules, respectively, were unclassifiable. Including unclassified malignant nodules (n = 1234, malignant = 50), comparison of the ROC curves showed lower performance of ATA [area under the curve (AUC) = ATA (0.49) vs. ACR TI-RADS (0.62), p = 0.008 and ATA vs. AACE/ACE/AME (0.59), p = 0.022]. Proportion of below size cutoff biopsies for ATA, ACR TI-RADS, and AACE/ACE/AME was different [16, 42, and 29% (all p < 0.001)], but no differences in malignancy rate were observed in these nodules. Conclusion: The present study is the first to validate in elderly patients these classifications showing that AACE/ACE/AME and ACR TI-RADS can predict thyroid malignancy more accurately than the ATA when unclassifiable malignant nodules are considered. Moreover, in this aged segment of the population, the use of ACR TI-RADS avoided more invasive procedures. Keywords: Elderly patients; Malignant cytology; Thyroid nodules; Ultrasound characteristics

Protein-protein-interaction network organization of the hypusine modification system

Hypusine modification of eukaryotic initiation factor 5A (eIF-5A) represents a unique and highly specific post-translational modification with regulatory functions in cancer, diabetes, and infectious diseases. However, the specific cellular pathways that are influenced by the hypusine modification remain largely unknown. To globally characterize eIF-5A and hypusine-dependent pathways, we used an approach that combines large-scale bioreactor cell culture with tandem affinity purification and mass spectrometry: "bioreactor-TAP-MS/MS." By applying this approach systematically to all four components of the hypusine modification system (eIF-5A1, eIF-5A2, DHS, and DOHH), we identified 248 interacting proteins as components of the cellular hypusine network, with diverse functions including regulation of translation, mRNA processing, DNA replication, and cell cycle regulation. Network analysis of this data set enabled us to provide a comprehensive overview of the protein-protein interaction landscape of the hypusine modification system. In addition, we validated the interaction of eIF-5A with some of the newly identified associated proteins in more detail. Our analysis has revealed numerous novel interactions, and thus provides a valuable resource for understanding how this crucial homeostatic signaling pathway affects different cellular functions

An atlas of genetic influences on human blood metabolites

Genome-wide association scans with high-throughput metabolic profiling provide unprecedented insights into how genetic variation influences metabolism and complex disease. Here we report the most comprehensive exploration of genetic loci influencing human metabolism thus far, comprising 7,824 adult individuals from 2 European population studies. We report genome-wide significant associations at 145 metabolic loci and their biochemical connectivity with more than 400 metabolites in human blood. We extensively characterize the resulting in vivo blueprint of metabolism in human blood by integrating it with information on gene expression, heritability and overlap with known loci for complex disorders, inborn errors of metabolism and pharmacological targets. We further developed a database and web-based resources for data mining and results visualization. Our findings provide new insights into the role of inherited variation in blood metabolic diversity and identify potential new opportunities for drug development and for understanding disease