SummaryDuring nephrogenesis, stem/progenitor cells differentiate and give rise to early nephron structures that segment to proximal and distal nephron cell types. Previously, we prospectively isolated progenitors from human fetal kidney (hFK) utilizing a combination of surface markers. However, upon culture nephron progenitors differentiated and could not be robustly maintained in vitro. Here, by culturing hFK in a modified medium used for in vitro growth of mouse nephron progenitors, and by dissection of NCAM+/CD133− progenitor cells according to EpCAM expression (NCAM+/CD133−/EpCAM−, NCAM+/CD133−/EpCAMdim, NCAM+/CD133−/EpCAMbright), we show at single-cell resolution a preservation of uninduced and induced cap mesenchyme as well as a transitioning mesenchymal-epithelial state. Concomitantly, differentiating and differentiated epithelial lineages are also maintained. In vitro expansion of discrete stages of early human nephrogenesis in nephron stem cell cultures may be used for drug screening on a full repertoire of developing kidney cells and for prospective isolation of mesenchymal or epithelial renal lineages for regenerative medicine.

In recent years, there has been an effort to develop new technologies for measuring gene expression and sequence information from thousands of individual cells. Large data sets that were obtained using these 'single cell' technologies have allowed scientists to address fundamental questions in biomedicine ranging from stems cells and development to cancer and immunology. Here, we provide a brief review of recent developments in single-cell technology. Our intention is to provide a quick background for newcomers to the field as well as a deeper description of some of the leading technologies to date.

The possibility of photoeradicating the prokaryotic microorganism Candida albicans by enhancing its endogenous porphyrin production and accumulation was investigated in this study. Induction of porphyrin synthesis was performed by the addition of delta-aminolevulinic acid (ALA), or its hydrophobic derivative ALA methyl ester (m-ALA). Photoinactivation of C. albicans was performed under blue light (407-420 nm) illumination. A decrease in viability of about 1.6 or 2.1 orders of magnitudes was obtained with a light dose of 36 J/cm(2) for an initial concentration of 100-mg/ml ALA or m-ALA, respectively. Endogenous porphyrins extracted from the cells showed that cultures incubated with m-ALA accumulated a relatively higher amount of endogenous porphyrins than ALA, indicating better transport through the yeast cell barriers. When a combination of miconazole and ketoconazole (antifungal agents) is given at a sub-inhibitory concentration (0.5 microg/ml each) with an inducer, a 2.1 or 3.2 orders of magnitude decrease in viability is caused with ALA or with m-ALA, respectively, upon illumination. Fluorescence intensities of the accumulated porphyrins as demonstrated by FACS indicate that the combination of the two azole drugs and an inducer cause a relatively high amount of endogenous porphyrins. Although the additive action of both azole drugs allow better penetration of the inducer, especially m-ALA photoeradication remained limited because of an acidic pH generated in the presence of the inducer. The acidic pH is probably the cause for the inefficiency of the photodynamic treatment. More hydrophobic inducers than m-ALA and less acidic must be investigated to improve the photodynamic treatment by endogenous-induced porphyrins.

During mammalian kidney development, nephron progenitors undergo a mesenchymal to epithelial transition and eventually differentiate into the various tubular segments of the nephron. Recently, the different cell types in the developing kidney were characterized using the Dropseq single cell RNA sequencing technology for measuring gene expression from thousands of individual cells. However, many genes can also be alternatively spliced and this creates an additional layer of heterogeneity. We therefore used full transcript length single-cell RNA sequencing to obtain the transcriptomes of 544 individual cells from mouse embryonic kidneys. We first used gene expression levels to identify each cell type. Then, we comprehensively characterized the splice isoform switching that occurs during the transition between mesenchymal and epithelial cellular states and identified several putative splicing regulators, including the genes Esrp1/2 and Rbfox1/2. We anticipate that these results will improve our understanding of the molecular mechanisms involved in kidney development.

The mechanism of photoinactivation of Candida albicans by 3.5 μM uncharged, cationic or anionic porphyrins under blue light (407-420 nm) was found to be dependent on the uptake of porphyrins into yeast cells, and was also dependent on the presence or absence of proteins in the photosensitization medium. In a very protein-rich medium, a decrease in viability was observed only with the uncharged porphyrin. Photoinactivation by uncharged or cationic porphyrins in a protein-poorer medium resulted in total eradication, whereas no significant decrease was observed with the anionic porphyrin. Phototreatment in PBS resulted in eradication with all three porphyrins. X-ray microanalysis after phototreatment by the uncharged or cationic porphyrins in the protein-poor medium exhibited ion loss, indicating cell-membrane damage. Transmission electron microscopy indicated cellular and chromosomal damage. No ion loss or cell damage was observed in this medium with the anionic porphyrin. The efficiency of photoeradication of C. albicans is dependent on porphyrin uptake, which might lead (upon illumination) to processes that facilitate the formation of reactive oxygen species that damage the cells. Uptake of charged porphyrins is dependent on protein quantity and quality in the photosensitization microenvironment. This fact must be taken into account when using charged photosensitizers.