iPSC-Derived Organoids as Therapeutic Models in Regenerative Medicine and Oncology.

Progress made during the last decade in stem cell biology allows currently an unprecedented potential to translate these advances into the clinical applications and to shape the future of regenerative medicine. Organoid technology is amongst these major developments, derived from primary tissues or more recently, from induced pluripotent stem cells (iPSC). The use of iPSC technology offers the possibility of cancer modeling especially in hereditary cancers with germline oncogenic mutations. Similarly, it has the advantage to be amenable to genome editing with introduction of specific oncogenic alterations using CRISPR-mediated gene editing. In the field of regenerative medicine, iPSC-derived organoids hold promise for the generation of future advanced therapeutic medicinal products (ATMP) for organ repair. Finally, it appears that they can be of highly useful experimental tools to determine cell targets of SARS-Cov-2 infections allowing to test anti-Covid drugs. Thus, with the possibilities of genomic editing and the development of new protocols for differentiation toward functional tissues, it is expected that iPSC-derived organoid technology will represent also a therapeutic tool in all areas of medicine.

Combinatory chemical and biological approaches to investigate metal elements in agricultural runoff water.

As part of a project studying the interactions between farming practices, soil erosion processes, and fate of agricultural pollutants into runoff waters, we conducted a pilot study to investigate the relationship between metal contents and metallothionein-2A (MT-2A) as a bioindicator of metal exposure. Runoff water samples were collected between May and November 1999 at the point of outlet of an elementary watershed located in the Paris basin. Selected metals (Al, As, Cd, Cr, Cu, Pb, Hg, Ni, and Zn) were analyzed using conventional techniques. In parallel, human T cells were exposed to water samples for 6 and 18 h and then cell viability and MT-2A gene expression were measured. Results show that among the 10 water samples tested, Al and Zn predominate (highest values = 4.9 and 2.6 microM, respectively), while other metals were below the microM level. Five out of 10 samples induced MT-2A gene expression (30-80% increase at 18 h) as compared with the control. When comparing MT-2A induction profile with metals contents, no obvious correlation was found, suggesting that additional components or parameters are involved. Finally, there was an apparent inverse relationship between Ca concentration and MT-2A gene induction. Although still preliminary, in the absence of longer monitoring, this study shows that MT-2A gene expression is a useful tool to complement chemical analysis in assessing metal elements in water. These combinatory approaches will be pursued and integrated in an ongoing watershed field research project.

Inhibition of in vitro cell adherence of Clostridium difficile by Saccharomyces boulardii.

The influence on the adherence of Clostridium difficile to Vero cells of the yeast Saccharomyces boulardii, the yeast fractions (cytoplasm and cell wall) and the culture supernatant was investigated in vitro. C. difficile adherence was significantly inhibited when bacteria were pre-incubated with the whole yeast and the cell wall fraction; this adherence inhibition was dose-dependent. The cell wall fraction also acts upon the target cultured cells inasmuch as the level of adherence was significantly decreased when Vero cells were preincubated with it. The same experiments carried out in the presence of an inhibitor of serine proteases resulted in no inhibition of bacterial adherence. These results suggest that the yeast could inhibit adherence of C. difficile to cells thanks to its proteolytic activity but also through steric hindrance.

A Clostridium difficile gene encoding flagellin.

Six strains of Clostridium difficile examined by electron microscopy were found to carry flagella. The flagella of these strains were extracted and the N-terminal sequences of the flagellin proteins were determined. Four of the strains carried the N-terminal sequence MRVNTNVSAL exhibiting up to 90% identity to numerous flagellins. Using degenerate primers based on the N-terminal sequence and the conserved C-terminal sequence of several flagellins, the gene encoding the flagellum subunit (fliC) was isolated and sequenced from two virulent strains. The two gene sequences exhibited 91% inter-strain identity. The gene consists of 870 nt encoding a protein of 290 amino acids with an estimated molecular mass of 31 kDa, while the extracted flagellin has an apparent molecular mass of 39 kDa on SDS-PAGE. The FliC protein displays a high degree of identity in the N- and C-terminal amino acids whereas the central region is variable. A second ORF is present downstream of fliC displaying homology to glycosyltransferases. The fliC gene was expressed in fusion with glutathione S-transferase, purified and a polyclonal monospecific antiserum was obtained. Flagella of C. difficile do not play a role in adherence, since the antiserum raised against the purified protein did not inhibit adherence to cultured cells. PCR-RFLP analysis of amplified flagellin gene products and Southern analysis revealed inter-strain heterogeneity; this could be useful for epidemiological and phylogenetic studies of this organism.