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In-vivo single cell clonal analysis in the adult mouse kidney has previously shown lineage-restricted clonal proliferation within varying nephron segments as a mechanism responsible for cell replacement and local regeneration. To analyze ex-vivo clonal growth, we now preformed limiting dilution to generate genuine clonal cultures from one single human renal epithelial cell, which can give rise to up to 3.4 * 106 cells, and analyzed their characteristics using transcriptomics. A comparison between clonal cultures revealed restriction to either proximal or distal kidney sub-lineages with distinct cellular and molecular characteristics; rapidly amplifying de-differentiated clones and a stably proliferating cuboidal epithelial-appearing clones, respectively. Furthermore, each showed distinct molecular features including cell-cycle, epithelial-mesenchymal transition, oxidative phosphorylation, BMP signaling pathway and cell surface markers. In addition, analysis of clonal versus bulk cultures show early clones to be more quiescent, with elevated expression of renal developmental genes and overall reduction in renal identity markers, but with an overlapping expression of nephron segment identifiers and multiple identity. Thus, ex-vivo clonal growth mimics the in-vivo situation displaying lineage-restricted precursor characteristics of mature renal cells. These data suggest that for reconstruction of varying renal lineages with human adult kidney based organoid technology and kidney regeneration ex-vivo, use of multiple heterogeneous precursors is warranted.

Summary During 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., Graphical Abstract, Highlights • mNPEM enables in vitro preservation of human renal embryonic CM and epithelia • EpCAM allows further dissection of expanded NCAM+CD133− early nephric population • Single-cell analysis unveils a continuous lineage hierarchy in nephrogenesis and WT • Splice isoform switching confirms a unified MET hierarchy in nephrogenesis and WT, In this article, Dekel and colleagues use flow cytometry and single-cell analysis to show that human fetal kidney cultured in modified nephron progenitor expansion medium (mNPEM) preserves all major nephric developmental lineages (including the Cap mesenchyme, early epithelial progeny, differentiated epithelium, and mesenchymal-to-epithelial-transition states). The biomarker system they developed allows distinguishing between early nephric lineages in expanded nephron stem cell cultures affording prospective isolation.

Nephrons are the functional units of the kidney. During kidney development, cells from the cap mesenchyme – a transient kidney-specific progenitor state – undergo a mesenchymal to epithelial transition (MET) and subsequently differentiate into the various epithelial cell types that create the tubular structures of the nephron. Faults in this transition can lead to a pediatric malignancy of the kidney called Wilms’ tumor that mimics normal kidney development. While kidney development has been characterized at the gene expression level, a comprehensive characterization of alternative splicing is lacking. We therefore performed RNA sequencing on cell populations representing early, intermediate, and late developmental stages of the human fetal kidney, as well as three blastemal-predominant Wilms’ tumor patient-derived xenografts. We identified a set of transcripts that are alternatively spliced between the different developmental stages. Moreover, we found that cells from the earliest developmental stage have a mesenchymal splice-isoform profile that is similar to that of blastemal-predominant Wilms’ tumors. RNA binding motif enrichment analysis suggests that the mRNA binding proteins ESRP1, ESRP2, RBFOX2, and QKI regulate mRNA splice isoform switching during human kidney development. These findings illuminate new molecular mechanisms involved in kidney development and pediatric kidney tumors.HIGHLIGHTSDuring fetal kidney development, kidney progenitor cells undergo a mesenchymal to epithelial transition (MET) and subsequently differentiate into the various epithelial cell types that create the tubular structures of the nephron.RNA sequencing identifies a set of transcripts that undergo splice isoform switching during the mesenchymal to epithelial transition (MET) that occurs in the course of human fetal kidney development.Cells in the early stages of kidney development have a mesenchymal splice-isoform profile that is similar to that observed in blastemal-predominant Wilms’ tumor patient-derived xenografts (WT-PDX) that represent an aggressive subtype of Wilms’ tumors.RNA binding motif enrichment analysis indicates that the mRNA binding proteins ESRP1, ESRP2, RBFOX2, and QKI regulate splice isoform switching during human kidney development.

In-vivo single cell clonal analysis in the adult mouse kidney has previously shown lineage-restricted clonal proliferation within varying nephron segments as a mechanism responsible for cell replacement and local regeneration. To analyze ex-vivo clonal growth, we now preformed limiting dilution to generate genuine clonal cultures from one single human renal epithelial cell, which can give rise to up to 3.4 * 10

Following reports of emotional psychopathology in children and adults exposed to organophosphates, the effects of postnatal chlorpyrifos (CPF) on fear-conditioning and depression-like behaviors were tested in adult mice. Concomitant changes in expression of mRNA for synaptic and soluble splice variants of acetylcholinesterase (AChE) were examined in mouse pups and adults of the Balb/C and C57Bl/6 (B6) strains, which differ in their behavioral and hormonal stress response. Mice were injected subcutaneously with 1 mg/kg CPF on postnatal days 4-10 and tested as adults for conditioned fear, sucrose preference, and forced swim. Acetylcholinesterase activity was assessed in the brains of pups on the first and last day of treatment. Expression of soluble and synaptic AChE mRNA was assessed in brains of treated pups and fear-conditioned adults using real-time PCR. Adult Balb/C mice exposed postnatally to CPF showed exacerbated fear-conditioning and impaired active avoidance. Adult B6 mice exposed postnatally to CPF showed a more specific fear response to tones and less freezing in the inter-tone intervals, in contrast to the vehicle-pretreated mice. Chlorpyrifos also attenuated sweet preference and enhanced climbing in the forced swim test. Chlorpyrifos-treated mice had increased expression of both synaptic and readthrough AChE transcripts in the hippocampus of Balb/C mice and decreased expression in the amygdala following fear-conditioning. In conclusion, postnatal CPF had long-term effects on fear and depression, as well as on expression of AChE mRNA. These changes may be related to alteration in the interaction between hippocampus and amygdala in regulating negative emotions.

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.

The role of the rare soluble splice variant of acetylcholinesterase (AChE-R) in anxiety behavior was assayed using the elevated plus maze (EPM). The effects of pretreatment with restraint stress and the acetylcholinesterase inhibitor diisopropylfluorophosphate (DFP) were tested, as these treatments are known to enhance the expression of AChE-R in several brain regions. Mice from the CD-1 outbred and C57BL/6 inbred strains were randomly assigned to seven treatment groups: homecage control, elevated plus maze without pretreatment, 3 days restraint stress or 3 days pretreatment with saline or one of three doses of DFP, for a total of 14 groups. All mice, except homecage controls, were tested twice on the elevated plus maze. AChE-R transcript expression was increased following elevated plus maze stress in hippocampus and amygdala, but not in the prefrontal cortex of CD-1 and not in C57BL/6 mice. Saline-injected C57BL/6 mice had reduced expression of AChE-R transcripts compared to untreated C57 BL/6 mice. DFP pretreatment reversed the stress-induced changes, increased AChE-R transcripts in CD-1 mice. AChE-R expression in the striatum and amygdala were positively correlated with anxiety in the EPM.

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.