Adipose tissue-derived mesenchymal stromal cells for clinical application: An efficient isolation approach.

PURPOSE OF THE STUDY: Mesenchymal stromal cells (MSCs) are considered a promising tool for cell therapy approaches. The translation of research-based cell culture protocols into procedures that comply with Good Manufacturing Practice (GMP) is critical. The aim of this study was to design a new method for the expansion of MSCs from Adipose Tissue (AT-MSCs) in compliance with GMP, without enzymatic tissue digestion and without the use of animal proteins as source of growth factors. PATIENTS AND METHODS: MSCs were expanded from 10 periumbilical biopsies. Our new isolation approach is based on: (1) disruption of AT with an automated, closed system; (2) use of GMP-grade medium without the addition of fetal bovine serum or platelet lysate; (3) use of human recombinant Trypsin. AT-MSCs cultured in α-MEM and minced by scalpel were used as control. RESULTS: It was possible to expand MSCs from all the AT-samples for at least eight passages. MSCs displayed the typical spindle-shape morphology, a high viability, multilineage differentiation potential and high expression levels of the typical MSC-specific surface antigens and genes. Compared to standard method, MSCs obtained with the new method showed higher yield, up to passage 6, and higher purity in terms of percentage of CD34 and CD45 markers. All AT-MSCs exhibit in vitro immunosuppressive capacity and possess a normal karyotype. CONCLUSIONS: Our data clearly demonstrate that our new approach permits to generate AT-MSCs fully compliant for therapeutic use and better at least in terms of quantity and purity than those obtained with the standard method.

p63, a p53 homologue, is a selective nuclear marker of myoepithelial cells of the human breast.

Myoepithelial cells (MCs) constitute the basal cell layer of normal mammary epithelia, and their identification is of particular diagnostic value because they are retained in most benign lesions while being lost in malignancy. Several MC immunocytochemical markers are currently available for diagnostic purposes, with special reference to smooth muscle-related antigens. p63 is a member of the p53 gene family, and its germline mutations are associated with severe mammary developmental defects in both rodents and humans. Different p63 isoforms have been identified, some of which (DeltaNp63) are preferentially expressed in the epithelial basal cells of different organs and have been considered as possible markers of stem cells/reserve cells. We investigated immunohistochemically 384 samples of normal and diseased human breast, including 300 invasive carcinomas, using four antibodies recognizing all p63 isoforms, or the DeltaNp63 isoforms. Twenty cytologic specimens were also investigated. Furthermore, snap-frozen tissue samples from three fibroadenomas and 10 invasive ductal carcinomas with their paired non-neoplastic tissues and three corresponding lymph node metastases were evaluated for the expression of p63 mRNA by RT-PCR. In normal breast tissue p63 immunoreactivity was confined to the nuclei of MCs. In all benign lesions p63-immunoreactive cells formed a continuous basal rim along the epithelial structures. Stromal cells, and in particular myofibroblasts, were consistently unreactive. Adenomyoepitheliomas showed nuclear staining in most neoplastic cells. A peripheral rim of p63-immunoreactive cells was retained surrounding lobular and ductal carcinoma in situ, although it was discontinuous as opposed to the normal structures. Invasive breast carcinomas were consistently devoid of nuclear p63 staining, with the exception of the two adenoid-cystic carcinomas, of the two ductal carcinomas with squamous metaplasia, and of 11 (4.6%) ductal carcinomas not otherwise specified, showing p63 immunoreactivity in a minor fraction (5-15%) of the neoplastic cells. In comparison with other MC markers, p63 was the most specific, being restricted exclusively to MCs, whereas antibodies to smooth muscle actin and, to a lesser extent, calponin also decorated stromal myofibroblasts. In the cytologic preparations p63 immunoreactivity was a consistent feature of "naked nuclei" and of a subset of cells surrounding benign epithelial clusters. RT-PCR experiments with primers specific for different p63 isoforms documented that normal tissues and fibroadenomas preferentially expressed the DeltaNp63 isoforms. Our study demonstrates that in normal and pathologic breast tissues MCs consistently express the DeltaNp63 isoforms. We suggest p63 as a reliable, highly specific, and sensitive MC marker in both histologic and cytologic preparations. Furthermore, because p63 immunoreactivity in adult epithelia is normally restricted to progenitor cells, it can be speculated that it might be a clue for the identification of the still elusive breast progenitor cells.

PAX8-PPARgamma1 fusion oncogene in human thyroid carcinoma [corrected].

Chromosomal translocations that encode fusion oncoproteins have been observed consistently in leukemias/lymphomas and sarcomas but not in carcinomas, the most common human cancers. Here, we report that t(2;3)(q13;p25), a translocation identified in a subset of human thyroid follicular carcinomas, results in fusion of the DNA binding domains of the thyroid transcription factor PAX8 to domains A to F of the peroxisome proliferator-activated receptor (PPAR) gamma1. PAX8-PPARgamma1 mRNA and protein were detected in 5 of 8 thyroid follicular carcinomas but not in 20 follicular adenomas, 10 papillary carcinomas, or 10 multinodular hyperplasias. PAX8-PPARgamma1 inhibited thiazolidinedione-induced transactivation by PPARgamma1 in a dominant negative manner. The experiments demonstrate an oncogenic role for PPARgamma and suggest that PAX8-PPARgamma1 may be useful in the diagnosis and treatment of thyroid carcinoma.

Small thymus in very low birth weight infants born to mothers with subclinical chorioamnionitis.

Chorioamnionitis, a major cause of preterm birth with significant neonatal morbidity and mortality, frequently occurs in mothers who are free of symptoms. A combined clinical, radiologic, and pathologic study of 129 very low birth weight infants indicated a significant association between a markedly decreased thymic size at birth and subclinical chorioamnionitis.

Relationship between mode of delivery and neonatal calcium homeostasis.

Serum levels of calcium (Ca), calcitonin (CT) and parathyroid hormone (PTH) were determined in cord blood of 229 newborns. In 136 newborns the tests were repeated 24 h later. The probands were divided into four groups according to mode of delivery: (1) spontaneous; (2) elective caesarean section without labour; (3) elective caesarean section in labour; (4) emergency caesarean section with fetal distress. Newborns in group 2 had significantly lower Ca and CT levels and significantly higher PTH concentrations in cord blood than the other three groups. In all groups Ca and PTH concentrations were negatively correlated. At 24 h, mean Ca levels had decreased and mean CT and PTH concentrations had increased in all four groups. Newborns in group 2 still had lower Ca levels but higher CT and PTH concentrations. At that time there were negative correlations between Ca and CT levels in groups 1 and 2 and between Ca and PTH concentrations in group 1. These data demonstrate that without labour, cord blood Ca and CT levels are lower and PTH concentrations are higher. The low 24 h calcium in newborns delivered without labour is explained by the lower Ca levels at birth and a tremendous increase of CT. The PTH secretion in full-term newborns is very substantial and negatively correlated with Ca levels.

[Distribution in a density gradient of human erythrocytes: sex-related differences]. / Distribuzione in gradiente di densita' degli eritrociti umani: differenze legate al sesso.

Differences in erythrocyte distribution on Ficoll-Sodium Metrizoate discontinuous density gradient were evaluated between adult males and females. Testosterone and androstenedione plasma levels were tested in males, oestradiol, progesterone and prolactin in both males and females. Female group showed a much higher proportion of lighter younger erythrocytes than males, while males showed higher proportion of denser older cells. Positive correlation was found between young cell percent and oestradiol level, both in males and females, but no correlation was found with androgen hormones.

[Changes in the density gradient distribution of erythrocytes during the menstrual cycle]. / Modificazioni della distribuzione in gradiente di densita' degli eritrociti durante il ciclo mestruale.

Modifications in erythrocyte density distribution were evaluated, by means of centrifugation on Ficoll-Triosil discontinuous density gradient, in five healthy young women during menstrual cycle. Oestradiol and progesterone plasma levels were tested, showing normal and typically ovulatory-like variations during the cycle. Youngest cell populations showed always quantitative increases from the follicular to the ovulatory phase of the cycle, reverting to basal values in the luteal phase. Possible hormonal or haemorrhagic causes are discussed.