Detection of PatIent-Level distances from single cell genomics and pathomics data with Optimal Transport (PILOT)

Although clinical applications represent the next challenge in single-cell genomics and digital pathology, we still lack computational methods to analyze single-cell or pathomics data to find sample-level trajectories or clusters associated with diseases. This remains challenging as single-cell/pathomics data are multi-scale, i.e., a sample is represented by clusters of cells/structures, and samples cannot be easily compared with each other. Here we propose PatIent Level analysis with Optimal Transport (PILOT). PILOT uses optimal transport to compute the Wasserstein distance between two individual single-cell samples. This allows us to perform unsupervised analysis at the sample level and uncover trajectories or cellular clusters associated with disease progression. We evaluate PILOT and competing approaches in single-cell genomics or pathomics studies involving various human diseases with up to 600 samples/patients and millions of cells or tissue structures. Our results demonstrate that PILOT detects disease-associated samples from large and complex single-cell or pathomics data. Moreover, PILOT provides a statistical approach to find changes in cell populations, gene expression, and tissue structures related to the trajectories or clusters supporting interpretation of predictions.</p

Datasheet1_Increased levels of a mycophenolic acid metabolite in patients with kidney failure negatively affect cardiomyocyte health.pdf

Chronic kidney disease (CKD) significantly increases cardiovascular risk and mortality, and the accumulation of uremic toxins in the circulation upon kidney failure contributes to this increased risk. We thus performed a screening for potential novel mediators of reduced cardiovascular health starting from dialysate obtained after hemodialysis of patients with CKD. The dialysate was gradually fractionated to increased purity using orthogonal chromatography steps, with each fraction screened for a potential negative impact on the metabolic activity of cardiomyocytes using a high-throughput MTT-assay, until ultimately a highly purified fraction with strong effects on cardiomyocyte health was retained. Mass spectrometry and nuclear magnetic resonance identified the metabolite mycophenolic acid-β-glucuronide (MPA-G) as a responsible substance. MPA-G is the main metabolite from the immunosuppressive agent MPA that is supplied in the form of mycophenolate mofetil (MMF) to patients in preparation for and after transplantation or for treatment of autoimmune and non-transplant kidney diseases. The adverse effect of MPA-G on cardiomyocytes was confirmed in vitro, reducing the overall metabolic activity and cellular respiration while increasing mitochondrial reactive oxygen species production in cardiomyocytes at concentrations detected in MMF-treated patients with failing kidney function. This study draws attention to the potential adverse effects of long-term high MMF dosing, specifically in patients with severely reduced kidney function already displaying a highly increased cardiovascular risk.</p

Expression of podocyte marker proteins by primary parietal or podocyte cell lines.

<p>A. Lysates of primary PECs or podocytes were subjected to immunoblotting for Pax2 expression. Primary PECs expressed significant amounts of Pax2, lower amounts were detected in primary podocytes (arrow). Lysates of endothelial cells (HUVEC or glomerular endothelial cells) were used as negative controls. B. After six passages, Pax2 was expressed in primary PECs and podocytes (arrow). Total kidney lysates were used as positive controls, endothelial cells were used as negative controls. C, C′ In immunofluorescent stainings on primary PECs and podocytes after six passages of culture, Pax2 was expressed in a nuclear fashion. Isotype-matched irrelevant antiserum was used as control (C′). D. Immunoblotting lysates of primary PECs or podocytes using ab sc192 showed that WT-1 is expressed in both cell types with significantly higher levels in primary podocytes (arrow). Lysates of endothelial cells were negative for WT1. E. After six passages, WT1 was expressed both in PECs and podocytes in a nuclear fashion (immunofluorescent staining using ab sc192). F. Parietal cells express low levels of WT1 also <i>in vivo</i> in mice with a mixed genetic background using ab sc846 (F, F′, arrowheads, immunohistological staining). For comparison, a strong expression of WT-1 was detected in podocytes (arrows). No WT-1 expression was noted elsewhere in the renal cortex, specifically not in proximal tubular cells (white arrowheads). G. Similarly, PECs expressed WT-1 also in other mouse genetic backgrounds (Sv129; WT-1/PAS staining) H, I. PECs (arrowheads) expressed low amounts of WT-1 also in normal Wistar rats (H) and humans (I). H′. Isotype matched controls were negative.</p

Generation of primary cultures of murine podocytes or parietal cells.

<p>A. First capsulated or decapsulated glomeruli were purified using magnetic beads (arrow with tails, >90% purity), to prepare primary mixed podocyte or parietal cell cultures respectively. For direct comparison, a capsulated glomerulus (arrows) is shown next to a decapsulated glomerulus (arrowheads). B, C. After 7–14 days of culture in EGM-MV media, primary outgrowths were obtained and stained for the genetic marker (X-gal). D, E. Next, suspensions of primary cells were treated with a luminescent substrate for beta-galactosidase (“labeled”, D′, E′) or not (controls, D, E) and were FACS-sorted using the indicated gate (“target cells”). In the example shown, 35,2% of the cells were defined as podocytes (D″) and 6.6% of the cells were defined as parietal cells (E″). F, G. The purity of enriched primary podocytes (F, F′) and parietal cells (G, G′) was evaluated after six passages in culture. Staining for the genetic marker beta-gal (X-gal) revealed that virtually all of the cells were either derived from podocytes or parietal cells (merged images: phase contrast+X-gal stainings). H, I. Similarly, in a FACscan analysis, virtually all cells were positive for the genetic tag beta-galactosidase (as revealed by metabolic labeling with FDG). In this representative analysis, 97.1% and 99.4% of the primary podocytes or PECs were defined as beta-gal positive, respectively.</p

Cell lineage tracing of cellular outgrowths.

<p>A–E. Outgrowths from isolated capsulated glomeruli of PEC-rtTA/LC1/R26R mice are predominantly derived from genetically tagged parietal cells. A. Genetic map of triple transgenic PEC-rtTA/LC1/R26R mice (cPodxl, rabbit podocalyxin promoter; rtTA, reverse tetracycline transactivator; Dox, doxycycline). B. Phase contrast image of cellular outgrowths from two capsulated glomeruli. C. Cellular outgrowths are derived from parietal cells as shown by X-gal staining (arrowhead). Within the glomerulus, magnetic beads used for isolation are still visible. D. Genetically tagged parietal cells at later time points (X-Gal staining, after 7 days of culture). E. After six passages, genetically tagged parietal cells show a spindle-shaped morphology (arrows; X-Gal crystals, arrowheads). F. A mixed culture of untagged primary podocytes and genetically tagged primary parietal cells was established to compare the morphology of the two cell types. Beta-gal-negative podocytes formed mostly larger cells (arrowheads) compared to beta-gal-positive parietal cells, which were mostly spindle-shaped (arrows, X-gal staining). G. Stills from time-lapse video (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034907#pone.0034907.s002" target="_blank">Video S1</a>, seconds indicated on lower right, 1 sec. = 75 min. capture time) of pure cultured parietal cells (see below). Arrowhead indicates a cell transitioning between spindle-shaped and polygonal morphology. H–J. Cellular outgrowths from decapsulated glomeruli of genetically tagged Pod-rtTA/LC1/R26R mice (after 7 or 10 days). H. Genetic map of the transgenic mice. I–J. In most cases, primary podocytes (blue) show a distinct morphology (larger cell body with prominent cytoplasm, arrowhead). For comparison, other cells without a genetic tag can be seen (J, arrow).</p

Transcriptome analysis of primary podocyte and PEC cultures.

<p>A. Eight transcriptomes were determined from two independent primary podocyte cultures and two independent primary PEC cultures grown in two different media (EGM-MV+20% FCS or RPMI+10% FCS), respectively. Transcriptomes were subjected to principal component analysis. PEC cultures (red spheres) are separated from podocyte cultures (blue spheres) along the axis of principle component 1 (PC1), which accounts for 47.8% of the overall variance in gene expression. Cells cultured in RPMI (light red and light blue spheres) segregate from those cultured in EGM-MV (bright red and bright blue spheres) along the axis of PC2, accounting for 11.8% of the variance. B. Primary podocyte and PEC cultures can also be clearly distinguished by cluster analysis of 2,507 significantly differentially regulated genes. C. Differential expression of the gene mRNA transcripts cadherin-3, EPH receptor A7, ladinin and scinderin in primary PEC cultures correlated with preferential expression in PECs versus podocytes in human kidney <i>in vivo</i> (arrowheads; images from <a href="http://www.proteinatlas.org" target="_blank">www.proteinatlas.org</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034907#pone.0034907-Uhlen1" target="_blank">[16]</a>). D. Expression levels of selected genes in primary podocyte and PEC cultures. Expression levels are given as mean logarithmic values to the base 2 of arbitrary intensity units.</p

Characterization of the primary PEC lines.

<p>PECs were negative for the endothelial marker <i>von Willebrand factor</i> (vWT, A) and for myofibroblast marker <i>alpha-SMA</i> (B). HUVECs (A′) or human dermal fibroblasts (B′) were used as positive controls. Polyclonal parietal cells were positive for claudin-1 (C) and caveolin-1 (D). C′, D′. Negative controls were performed using isotype-matched irrelevant primary antibodies. E. A significantly lower expression of synaptopodin was observed in parietal cells expressed even after six passages compared to primary podocytes (E′) or an immortalized podocyte cell line IHPC (E″). The findings were confirmed by SDS-page with subsequent immunoblotting using lysates of parietal cell cultures 2, 8 and 11 (F–H). Lysates of polyclonal primary podocyte cultures as well as of an immortalized podocyte cell line IHPC also showed expression of claudin-1 and calveolin-1. No expression of caveolin-1 was observed in Hel-1, GDM or Set-1 cell lines (G). Equal loading was verified by Ponceau S stain. H. Podocin expression is absent in primary PECs and down regulated in primary podocyte cells and in an immortalized podocyte cell line IHPC. I. Podoplanin is differentially expressed in primary podocytes relative to primary PECs after six passages in culture (arrow), consistent with mRNA expression analysis. Lysates of isolated glomeruli are used as positive control (H, I).</p