Multipotent adult rat, thyroid stem cells can be differentiated to follicular thyrocyte, and hepatocyte- like cells in 2D and 3D culture systems

We have recently characterized and differentiated towards endodermal and mesoder- mal lineages progenitor cells of the adult rat thyroid, expressing multipotency markers [1]. We have now assessed their clonogenicity, extent of side population, consistency of stem cell marker expression, and commitment to either follicular or hepatocyte-like lineages when in monolayer (2D), and suspension or Matrigel (3D). Colony forming unit (CFU)-like cultures were obtained by long-term subcultures of primary rat thyroid cells, under starvation conditions. CFU-like cultures seeded in Petri dishes by limiting dilution (1 cell / cm2) were observed to give rise to toluidine blue-positive, individual clones. In these cultures, quantitative densitometric analysis of immunoblotted Oct-3/4, Sca1, and GATA4 revealed an increase in stem cell markers ranging from 95% to 270% with respect to standard, primary thyroid cultures. In addition, using three different analytical techniques including DyeCycle Violet staining by flow cytometry, ABCG2 immunocytochemistry, and Hoechst 33342 histochemistry + the ABCG2 inhibitor, verapamil a side population involving 1-2% of CFU-like cultures was detected. Then, CFU-like cultures were differentiated using TSH, either in 2D or in 3D. Differentiated adherent cells resulted immunopositive for thyrocyte markers including thyroglobulin (TG), sodium-iodide symporter (NIS), and thyroperoxidase (TPO). Differentiation in suspension and in Matrigel gave rise to follicles with cells having ultrastructural features consistent with thyrocytes, and immunoreactivity (IR) for TG, NIS, and TPO. Finally, CFU-like cultures were differentiated in adherence to hepatocyte-like cells, resulting in pre-hepatocyte morphology, high periodic acid-Schiff reaction, and IR for α-fetoprotein and albumin. We conclude that our CFU-like thyroid cultures are enriched with a multipotent, stem cell population whose hepatic differentiation capacity has been revealed for the first time

Abdominal aorta aneurysm with hostile neck: Early outcomes in outside instruction for use in patients using the treovance® stent graft

Purpose: The efficacy and safety of endovascular aneurysm repair (EVAR), in patients outside instruction for use (IFU), is very challenging and widely debated. The aim of this study was to evaluate the placement of the Treovance® abdominal aorta stent-graft in patients with hostile proximal necks considered outside IFU. Materials and Methods: Between May 2013 and August 2014, 5 patients with outside IFU underwent EVAR with the Treovance® stent-graft. Technical and clinical successes were evaluated. All 5 patients underwent clinical and imaging follow-up. Results: Technical and clinical successes were achieved in all 5 patients without adjunctive endovascular procedures or surgical conversion. During the mean follow-up of 21 months, no type I/III endoleaks, stent-graft migration nor kinking/occlusion were observed. In all 5 patients, a reduction of the proximal neck angle was observed. Conclusion: In our small series of selected outside IFU patients, EVAR with the Treovance® stent-graft was technically feasible and safe, with satisfactory short-term follow-up results, when performed by experienced operators. Long-term follow-up will be necessary to confirm the durability of our preliminary promising results

Gli organi endocrini bioartificiali: prospettive della ricerca traslazionale applicata alla medicina rigenerativa in endocrinologia

Ricostruire in laboratorio, ossia ex situ, ghiandole endocrine bioartificiali è una prospettiva innovativa della ricerca traslazionale applicata alla medicina rigenerativa dei disturbi endocrino-metabolici. Utilizzando cellule staminali o primarie è possibile ingegnerizzare organoidi funzionali mediante ricellularizzazione sia di matrici tridimensionali (3D) acellulari allogeniche o xenogeniche, derivate da una ghiandola endocrina come quella che si vuole riprodurre, sia supporti reticolari 3D biocompatibili amorfi o che mimano la morfologia originaria delle ghiandola, cioè organomorfi. Con questo metodo sono stati ricostruiti in modelli animali insule pancreatiche, follicoli ovarici e tiroidei, tubuli seminiferi del testicolo e parte della corteccia surrenale. Utilizzando cellule umane è stato riprodotto un abbozzo di ovaio e microaggregati di paratiroide. Le ghiandole endocrine bioartificiali promettono di fornire un’alternativa alla terapia sostitutiva con ormoni di sintesi, che richiede compliance da parte del paziente, integrandosi nei circuiti naturali di feed-back. Inoltre, quando generate con cellule autologhe e supporti organomorfi individualizzati, è plausibile riproducano una condizione secretiva simile a quella originale del soggetto donatore (terapia personalizzata). Infine, il loro uso potrebbe ridurre i costi sostenuti dal Servizio Sanitario Nazionale per le terapie croniche, superando i limiti etici e farmacologici connessi al trapianto cellulare / tissutale da cadavere e donatore vivente allogenico o xenogenico e promuovendo il mercato della salute attraverso lo sviluppo delle biotecnologie mediche, analogamente a quanto accadde, oltre un trentennio addietro, per quello delle tecnologie informatiche, con l’avvento dei semiconduttori e dei calcolatori elettronici

Thyrogenic, adipogenic, and osteogenic differentiation of adult rat, thyroid stem cells enriched by long-term adherent subculture

We recently identified adult progenitor cells expressing multipotency markers in the rat thyroid (1). We have now studied these markers in primary cultures, thyrospheres, and adherent cells exhibiting features of side population / multilineage differentiation. Primary rat thyroid monolayers were immunolabeled / immunoblotted for ABCG2, Oct-3/4, HNF4 and Sca1. Thyrospheres were cytospinned and immunolabeled for Oct-3/4. Long-term subcultures were obtained by re-seeding monolayers at very low density, and growing them up to 5 months, using a starvation protocol to obtain colony forming unit (CFU)-like cultures. The latter were incubated with Hoechst (Hch) 33342 + the ABCG2 inhibitor, verapamil (VE), to identify a side population, and immunostained for ABCG2, vimentin (VIM), and cytokeratin (CYT). Thyroid monolayers and CFU-like cultures were differentiated using TSH, adipogenic, and osteogenic media. Up to 1/4 cells from primary monolayers and thyrospheres resulted either ABCG2-, Oct-3/4-, HNF4-or Sca-1-positive. In contrast, in CFU-like cultures ABCG2 was detected in up to 1/3 cells, whereas VIM was ubiquitous, and CYT disappeared. Consistently, a side population was revealed by the Hch-VE staining. Finally, CFU-like cultures differentiated to cells containing either thyroglobulin, or red oil-, or alizarin red-positive deposits. We conclude that multilineage differentiation of our CFU-like thyroid cultures reveals enrichment of a thyroid stem cell population

TOWARDS ADDITIVE MANUFACTURING OF RAMIFIED SCAFFOLDS OF THE THYROID VASCULAR SYSTEM: A PRELIMINARY FRACTAL ANALYSIS

Fractal properties have been demonstrated in literature for several human vascular systems. In the frame of the investigation of additive manufacturing (AM) as a viable solution to prototype single arterial branches of human soft tissue organs, the paper provides a fractal analysis of the arterial tree of the human thyroid gland. The possibility that the thyroid arterial structure may be described as auto-similar is investigated, by studying injection-corrosion casts of the cadaveric gland. Vessel branching is analyzed by measuring branch diameters, ramification angles, and vessel lengths with the use of an optical microscope. Metrological results are made dimensionless by applying, as a scaling parameter, the caliber of major arteries. Data are then studied on a cumulative basis and processed to infer general rules for vessel branching. High resolution microtomography (mCT) is used to determine the spaces occupied by vascular branches and calculate their planar fractal dimension. Finally, the vascular tree has been simulated by a mixed, stochastic / deterministic algorithm based on diffusion limited aggregation (DLA), in which mean values of vascular variables are set as constraints. The purpose of this research is to understand if fractality can be reliably assumed for computational modeling of the organ anatomy, in order to be able to produce, by AM, more representative physical prototypes and scaffolds. The finding allow to affirm that the human thyroid arterial structure exhibits a degree of auto similarity

Endocanalicular transendothelial crossing (ETC): A novel intravasation mode used by HEK-EBNA293-VEGF-D cells during the metastatic process in a xenograft model.

In cancer metastasis, intravasation of the invasive tumor cell (TCi) represents one of the most relevant events. During the last years, models regarding cancer cell intravasation have been proposed, such as the "endocanalicular transendothelial crossing" (ETC) theory. This theory describes the interplay between two adjacent endothelial cells and the TCi or a leukocyte during intravasation. Two endothelial cells create a channel with their cell membranes, in which the cell fits in without involving endothelial cell intercellular junctions, reaching the lumen through a transendothelial passage. In the present study, ten SCID mice were subcutaneously xenotransplanted with the HEK-EBNA293-VEGF-D cell line and euthanized after 35 days. Post-mortem examinations were performed and proper specimens from tumors were collected. Routine histology and immunohistochemistry for Ki-67, pAKT, pERK, ZEB-1, TWIST-1, F-actin, E-cadherin and LYVE-1 were performed followed by ultrastructural serial sections analysis. A novel experimental approach involving Computed Tomography (CT) combined with 3D digital model reconstruction was employed. The analysis of activated transcription factors supports that tumor cells at the periphery potentially underwent an epithelial-to-mesenchymal transition (EMT)-like process. Topographical analysis of LYVE-1 immunolabeled lymphatics revealed a peritumoral localisation. TEM investigations of the lymphatic vessels combined with 3D digital modelling enhanced the understanding of the endotheliocytes behavior during TCi intravasation, clarifying the ETC theory. Serial ultrastructural analysis performed within tumor periphery revealed numerous cells during the ETC process. Furthermore, this study demonstrates that ETC is an intravasation mode more frequently used by the TCi than by leukocytes during intravasation in the HEK-EBNA293-VEGF-D xenograft model and lays down the potential basis for promising future studies regarding intravasation blocking therapy

A planar fractal analysis of the arterial tree of the human thyroid gland: implications for additive manufacturing of 3D ramified scaffolds

It is currently known that a number of human vascular systems have a fractal geometry. Since we have recently developed a technique to prototype single arterial branches of human soft tissue organs by additive layer manufacturing (AM), we have explored the possibility that auto-similarity in vessel branching represents a key variable for accurate computational modeling of the organ three-dimensional (3D), macro / microscopic anatomy, and its reproduction by inverse engineering. To this purpose, ramification features of the intra-lobar arteries of the human thyroid were studied using injection-corrosion casts of a cadaveric gland. Vessel diameters, ramification angles and branch lengths were measured by light microscopic, computer-aided optical metrology. Distribution of morphological variables was considered on a cumulative basis, and special focus was given to the branching laws. To reduce the bias of vascular distortion due to the pressure of intra-vascular resin injection, measures were made dimensionless through the use of a scaling parameter set on the vascular caliber of major afferent arteries. In addition, using high resolution micro-tomography (mCT Skyscan 1172, Bruker micro-CT) equipped with CTAn software and the Otsu algorithm for segmentation, spaces occupied by vascular branches (referred to as Volume of Interests, VOI) were selected, and their planar fractal dimension calculated (Mandelbrot 1982). Finally, a computational simulation of the vascular tree was achieved using a mixed, stochastic / deterministic algorithm, based on diffusion limited aggregation (DLA; Witten and Sander, 1981), constrained by mean values of vascular variables. Ratios among decreasing cast calibers, ramification angles and branch lengths, respectively, were found strictly interrelated, mCT-VOI depicted fractal dimensions, and DLA simulation led to a fractal-like organization consistent with real data morphometrics. In summary, thyroid arterial geometry reliably exhibited a degree of auto-similarity, suggesting that fractality is a key feature for computational modeling and eventual AM of 3D vascular networks of the human thyroid