Salvatore Mosca

Anthropological Variations in the Anatomy of the Human Thyroid Arteries

Knowledge of anatomic variability of the superior (STA), inferior (ITA), and lowest accessory (IMA) thyroid arteries may be helpful in certain clinical conditions. However, details of this variability have not been thoroughly described. Specifically, whether the presence and site of origin of STA, ITA, and IMA are influenced by the anthropological group, to what extent their origin is symmetric or asymmetric, and the role played by this variability in visualizing each thyroid artery by nonselective thyroid angiography is not known. To clarify this we conducted a meta-analytical study on Caucasian and Asian subjects, the latter including only Japanese and Koreans. In Caucasians and Asians the presence of superior vessels compared to inferior vessels was more frequent and the probability of symmetric or asymmetric arterial origin for STA were equivalent. However, better recognition of inferior rather than superior vessels was achieved by nonselective angiography in Caucasians. Finally, different frequencies of presence and site of origin for each artery were identified in Caucasians compared to Asians. Our results suggest that the higher frequency of IMA in Asians than in Caucasians should result in a search for an IMA-dependent feeding artery of inferior parathyroid adenomas, primarily the mediastinal ones, especially in Asians both by imaging and transcatheter ablative approaches. In addition, we have found that a small percentage of Caucasian subjects lack an STA on the left side. Therefore, anatomic arterial compatibility should be carefully evaluated in the preoperative stage of laryngeal transplantation maintaining in situ the donors thyroid by terminal anastomoses between donor and recipient STAs. Finally, the lack of any individual thyroid artery in either Caucasians or Asians might influence the distribution of autonomic supply that runs with thyroid vessels to the thyroid parenchyma. This appears functionally relevant in cases of traumatic or surgical lesions of the cervical sympathetic chain involving thyroid nerves. In fact, a restricted local autonomic control of thyroid activity might be related to individual rami of thyroid nerves.

Effect of hypothyroidism on vasoactive intestinal polypeptide-immunoreactive neurons in forebrain-neurohypophysial nuclei of the rat brain

We have recently reported that hypothyroidism increases immunoreactive (IR)-vasoactive intestinal polypeptide (VIP) and VIP mRNA content in both parvocellular and magnocellular neurons of the rat, hypothalamic paraventricular nucleus (PVN). As VIP can stimulate vasopressin (AVP) secretion, we conducted an anatomical investigation to determine whether VIP-containing neurons in other regions of the brain that are involved with homeostatic mechanisms of water and salt conservation are also affected by hypothyroidism. The distribution and intensity of VIP immunostaining in neurons and fibers of the magnocellular-neurohypophysial system, including the hypothalamic PVN, supraoptic nucleus (SON) and accessory magnocellular cell groups, circumventricular subfornical organ (SFO), preoptic and anterior hypothalamus, midline thalamus, subthalamic zona incerta and posterior septal nuclei were studied using a highly sensitive immunocytochemical technique and unbiased neuronal counting methods, based on the optical dissector principle. Hypothyroidism increased the intensity of VIP immunostaining and/or the number/section, percentage and numerical density of IR-VIP neurons in the PVN, SON, nucleus circularis, periventricular preoptic nucleus of the hypothalamus and SFO. In addition, IR-VIP perikarya and/or fibers in the hypothalamic medial preoptic area and anterior periventricular nucleus, nucleus reuniens of the thalamus and dorsal fornix-triangular septal nucleus complex were also apparent in the hypothyroid animals while no immunostaining was seen in these areas in control animals. No quantitative and/or qualitative modifications in IR-VIP neurons and fibers were noted in the anterior hypothalamic area, suprachiasmatic nucleus, thalamic paraventricular nucles an subthalamic zona incerta between hypothyroid and control animals. These findings suggest an inverse relationship between thyroid hormone and VIP content and/or distribution of IR-VIP neurons in specific forebrain regions involved in the control of AVP release, extracellular fluid volume, thirst, blood pressure and anterior pituitary secretion. This raises the possibility that changes in fluid homeostasis and cardiovascular function occurring in hypothyroidism may be mediated, at least in part, by VIP-producing neurons in diverse regions of the brain.

Growth on poly(l-lactic acid) porous scaffold preserves CD73 and CD90 immunophenotype markers of rat bone marrow mesenchymal stromal cells

Few data are available on the effect of biomaterials on surface antigens of mammalian bone marrow-derived, adult mesenchymal stromal cells (MSCs). Since poly(l-lactic acid) or PLLA is largely used in tissue engineering of human bones, and we are developing a reverse engineering program to prototype with biomaterials the vascular architecture of bones for their bioartificial reconstruction, both in humans and animal models, we have studied the effect of porous, flat and smooth PLLA scaffolds on the immunophenotype of in vitro grown, rat MSCs in the absence of any coating, co-polymeric enrichment, and differentiation stimuli. Similar to controls on plastic, we show that our PLLA scaffold does not modify the distribution of some surface markers in rat MSCs. In particular, the maintained expression of CD73 and CD90 on two different subpopulations (small and large cells) is consistent with their adhesion to the PLLA scaffold through specialized appendages, and to their prominent content in actin. In addition, our PLLA scaffold favours retention of the intermediate filament desmin, believed a putative marker of undifferentiated state. Finally, it preserves all rat MSCs morphotypes, and allows for their survival, adhesion to the substrate, and replication. Remarkably, a subpopulation of rat MSCs grown on our PLLA scaffold exhibited formation of membrane protrusions of uncertain significance, although in a size range and morphology compatible with either motility blebs or shedding vesicles. In summary, our PLLA scaffold has no detrimental effect on a number of features of rat MSCs, primarily the expression of CD73 and CD90.

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, HNF4a 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-, HNF4a-, 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.

Adult stem / progenitor cells of the rat thyroid: side population distribution, intermediate filament expression, and long-term in vitro expansion

We recently identified adult stem / progenitor cells in the male rat thyroid, based on expression of the multipotency maker, ATP-binding cassette subfamily G member 2 (ABCG2) (1). To characterize these cells, we have now determined their distribution as a side population of the adult thyroid gland, identified their epithelial vs mesenchymal commitment by the presence of cytoplasmic intermediate filaments, and enriched their number using long-term, in vitro expansion of adherent elements. Sprague-Dawley male rats (50-75 gr) were used as thyroid donors. Following penthobarbital anesthesia rats were thyroidectomised, and primary cells prepared using enzymatic breaking of the gland. After 72 hs in standard monolayer culture, adherent cells were trypsinized, and either incubated for 90 min with the vital dye, Hoechst 33342 (Hch) + the ABCG2 inhibitor, verapamil (VE, 150 mM) followed by cytospin (1300 RPM x 8 min) for single, double, and triple light microscopic immunocytochemistry (IC), or re-seeded (20 x103 / cm2) in monolayer and grown up to 4 months, using a starvation protocol based on a single weekly change of culture medium (low glucose DMEM / 15 % FBS-FHS serum). Co-localization of nuclear Hoch with immunoreactive (IR) ABCG2 (rabbit anti-human polyclonal antiserum, 1:300), IR-cytokeratin (CTK, mAb 1:200), and IR-vimentin (VIM, mAb 1:100) was assessed by the ABC and indirect fluorescence techniques, using DAB, FITC and TRITC as chromogens. Rat kidney, human keratinocyte cell line, NTCT 2544 (courtesy of C. Pellegrini), and primary mouse and human fibroblasts (courtesy of D. Mattioli) were used as positive controls for IC. A consistent increase in Hch-positive nuclei was observed in VE-treated cultures, as opposed to VE-untreaded monolayers. In addition, an inverse staining relationship occurred between nuclear Hch and IR-CTK, as opposed to a direct relationship between nuclear Hch and IR-VIM. Co-localization of IR-ABCG2 with IR-CTK was seen in some cells, whereas that of IR-ABCG2 with IR-VIM was only occasionally detected. Finally, longterm expansion of primary thyrocytes resulted in 30% increase in IR-ABCG2 cells, as opposed to less than 1% IR-ABCG2 elements in standard culture. We conclude that ABCG2-positive cells of the rat thyroid are a side population of stem / progenitor elements, they are primarily committed to the epithelial phenotype, and can be enriched in vitro as adherent cells, suggesting clonal expansion.

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.