Urine-derived cells provide a readily accessible cell type for feeder-free mRNA reprogramming.

Over a decade after their discovery, induced pluripotent stem cells (iPSCs) have become a major biological model. The iPSC technology allows generation of pluripotent stem cells from somatic cells bearing any genomic background. The challenge ahead of us is to translate human iPSCs (hiPSCs) protocols into clinical treatment. To do so, we need to improve the quality of hiPSCs produced. In this study we report the reprogramming of multiple patient urine-derived cell lines with mRNA reprogramming, which, to date, is one of the fastest and most faithful reprogramming method. We show that mRNA reprogramming efficiently generates hiPSCs from urine-derived cells. Moreover, we were able to generate feeder-free bulk hiPSCs lines that did not display genomic abnormalities. Altogether, this reprogramming method will contribute to accelerating the translation of hiPSCs to therapeutic applications.

Osteopontin is an argentophilic protein in the bone matrix and in cells of kidney convoluted tubules.

Nucleolar organising regions (NOR) are part of the nucleolus, containing argyrophilic proteins (nucleoclin/C23, nucleophosmin/B23). They are identified by silver staining at low pH. The method also reveals osteocyte canaliculi and cement lines and granules in the cytoplasm of kidney cells in locations that mimic osteopontin distribution. Human bone and kidney sections, benign and lymphomatous pleural effusions were processed for silver staining to identify AgNOR. Sections were processed in parallel for immunohistochemistry with an antibody direct against osteopontin. In pleural effusions, AgNORs were found increased in the nuclei of lymphoma cells. In bone, Ag staining identified AgNOR in cell nuclei, as well as in osteocyte canaliculi, cement and resting lines. In the distal convoluted tubules of the kidney, silver deposits were also observed in cytoplasmic granules on the apical side of the cells. Immunolocalization of osteopontin closely matched with all these locations in bone and kidney. NOR proteins and osteopontin are proteins containing aspartic acid rich repeats that can bind Ag. Staining protocols using silver nitrate at low pH can identify these proteins on histological sections. AgNOR is a useful histochemical method to identify osteopontin in bone sections.

Microcomputed tomography used in the analysis of the morphology of root canals in extracted wisdom teeth.

Microcomputed tomography is a new technique for the non-destructive study of porous biological materials. We examined 11 wisdom teeth that had been removed prophylactically by microcomputed tomography operating in the cone beam mode. The two-dimensional sections of the teeth were reconstructed with surface rendering software to provide three-dimensional models that were observed and handled in virtual reality. The tooth itself, or the pulp chamber and canals, can be reconstructed and observed separately or simultaneously. Many teeth looked dystrophic with abnormal distributions of roots and canals. Microcomputed tomography seems to be a promising way of studying dental anatomy.

Increased nucleolar organizer regions in osteoclast nuclei of Paget's bone disease.

The etiology of Paget's disease of bone is still unknown but several studies have reported a viral origin. At the electron microscopic level, characteristic nuclear and cytoplasmic inclusions have been found and mimic paramyxoviral nucleocapsids in osteoclasts (Oc). Sarcomatous degeneration is observed in 2% of pagetic patients. Nuclear organizer regions are parts of the nucleolus involved in the synthesis of ribosomes, and they contain nonhistone proteins that can be silver stained (AgNORs) on the interphasic nuclei. AgNOR number is known to correlate with the proliferative activity of the cell populations, whether normal or malignant. Cancer cells have an increased demand for (robosomal) rRNA and correlations have been found between AgNORs and proliferative antigens. We have adapted the AgNOR staining method to undecalcified bone biopsies at the light and TEM levels. Bone sections from 10 pagetic patients (without a previous bisphosphonate treatment) were stained for AgNORs. 10 patients having metabolic bone diseases associated with an increased Oc number (i.e., primary and secondary hyperparathyroidism) were used as controls. AgNORs appeared as black dots within the nucleoli of Oc nuclei and were easily numbered. A maximum of two or three dots could be seen in Oc nuclei from control subjects. In pagetic Oc, AgNOR number was greatly increased (6.80 +/- 2.57 dots vs. 2.12 +/- 1.07 in controls). TEM study also showed AgNOR in the nucleoli of pagetic patients' Oc. The viral inclusions within the nuclei appeared faintly stained and could not be confused with AgNORs. The large number of AgNORs in the nuclei of pagetic Oc reflects the need for a greater abundance of ribosomes. With the pagetic Oc being highly active, the cytoplasmic synthesis of proteins is maximized (probably hydrolases involved in the matrix breakdown). An increase in AgNORs does not reflect the proliferative activity of the cell because Oc are made by the fusion of precursors. It is postulated that: (a) other mRNAs (of viral/oncogene origin) could be actively transcribed in pagetic patients and require more numerous ribosomes; or (b) a viral genome/oncogene promotes alteration of the nuclear/nucleolar mechanism.

Spectral and lifetime fluorescence imaging microscopies: new modalities of multiphoton microscopy applied to tissue or cell engineering.

Spectral and multiphoton imaging is the preferred approach for non-invasive study allowing deeper penetration to image molecular processes in living cells. But currently available fluorescence microscopic techniques based on fluorescence intensity, such as confocal or multiphoton excitation, cannot provide detailed quantitative information about the dynamic of complex cellular structure (molecular interaction). Due to the variation of the probe concentration, photostability, cross-talking, its effects cannot be distinguished in simple intensity images. Therefore, Time Resolved fluorescence image is required to investigate molecular interactions in biological systems. Fluorescence lifetimes are generally absolute, sensitive to environment, independent of the concentration of the probe and allow the use of probes with overlapping spectra but that not have the same fluorescence lifetime. In this work, we present the possibilities that are opened up by Fluorescence Lifetime Imaging Microscopy, firstly to collect images based on fluorescence lifetime contrast of GFP variants used as a reporter of gene expression in chondrocytes and secondly, to measure molecular proximity in erythrocyte (glycophorin/membrane) by Fluorescence Resonance Energy Transfer (FLIM-FRET).

Non-connected versus interconnected macroporosity in poly(2-hydroxyethyl methacrylate) polymers. An X-ray microtomographic and histomorphometric study.

Poly(2-hydroxyethyl methacrylate) (pHEMA) has potentially broad biomedical applications: it is biocompatible and has a hardness comparable to bone when bulk polymerized. Porous biomaterials allow bone integration to be increased, especially when the pores are interconnected. In this study, three types of porogens (sugar fibers, sucrose crystals, and urea beads) have been used to prepare macroporous pHEMA. The pore volume and interconnectivity parameters of the porosity were measured by X-ray microtomography and image analysis. Sucrose crystals, having a high volumetric mass, gave large pores that were located on the block sides. Urea beads and sugar fibers provided pores with the same star volume (2.65 +/- 0.46 mm3 and 2.48 +/- 0.52 mm3, respectively) but which differed in interconnectivity index, fractal dimension, and Euler-Poincarés number. Urea beads caused non-connected porosity, while sugar fibers created a dense labyrinth within the polymer. Interconnectivity was proved by carrying out surface treatment of the pHEMA (carboxymethylation in water), followed by von Kossà staining, which detected the carboxylic groups. Carboxymethylated surfaces were observed on the sides of the blocks and on the opened or interconnected pores. The disconnected pores were unstained. Macroporous polymers can be prepared with water-soluble porogens. X-ray microtomography appears a useful tool to measure porosity and interconnectedness.