Project description:Purpose: Bulk transcriptomics analysis of Wilms tumor SIX2+CITED1+ cells to compare and identify unique nephron progenitor transcriptome profiling (RNA-seq) signature between unfavorable and favorable Wilms Tumors and against human fetal kidney Methods: Wilms tumor and human fetal kidney samples were collected and transported on ice at 4°C in RPMI-1640 and a single cell suspensions were prepared following mechanical and enzymatic dissociation as previously publication. Enzymatic dissociation was performed with 125 U/ml collagenase I in RPMI-1640 at 37 °C for 35 min. The digested cells were then passed through a 100-μm cell strainer and a 40-μm cell strainer with washes of 1x PBS. The cell suspension was than centrifuged at 1500 rpm for 5 min and erythrocytes were eliminated using a red blood cell lysis kit. WT SIX2+CITED1+ cells were isolated using SIX2-Cy5 and CITED1-Cy3 Smartflare RNA probes following manufacturer’s instructions and previous publication. Briefly, cells were incubated over night at 37 °C with both RNA probes diluted at 1:20 in PBS and 25ul/ml in RPMI-1640 supplemented with 5% FBS, and 0.2% antimicrobial agent Primocin. After 16-18 h, cells were dissociation using TrypLE 1x for 5 min, cells were centrifuged at 1500 rpm for 5 min and prepared for FACS. Freshly isolated SIX2+CITED1+ cells from a 16 WGA hFK, freshly isolated SIX2+CITED1+ cells from WT8 (favorable stage II Wilms’ Tumor), freshly digested xenograft derived from cultured SIX2+CITED1+ cells (Passage 6) isolated from WT8, freshly digested xenograft generated from freshly isolated SIX2+CITED1+ cells from WT8, and total cell population of WT8. Approximately 3,000 cells were captured on a 10x Chromium device using a 10X V2 Single Cell 3′ Solution kit. All protocols were performed following manufacturer's instructions. Final library concentrations were determined using a Qubit high Sensitivity DNA assay Kit. Final sequencing libraries were analyzed on a Sequencing: HiSeq 4000, 2x150bp PE, and ~625M total reads/lane (QuickBiology) to determine the library size; Approximately, 300 million reads per sample were sequenced Results: Transcriptomics analysis of Wilms Tumor SIX2+CITED1+ cells in comparison to human fetal kidneys confirmed the nephrogenic signature of hFK-SIX2+CITED1+ and WT-SIX2+CITED1+ cells but highlighted differences in expression of pluripotency and self renewal-related genes.Trajectory inference analysis generated from sc-RNAseq data of SIX2+CITED1+ cells and cells dissociated from total WT (from where the SIX2+CITED1+ cells were isolated), suggests that SIX2+CITED1+ cells are the root cells that give rise to all the tumor cell types. Conclusion: Our study represents the first single cell transcriptomic characterization of Wilms Tumor cancer stem cells (SIX2+CITED1+) against human fetal kidney SIX2+CITED1+ cells and against the total tumor and xenografts from cultured and fresh isolated SIX2+CITED1+ WT cells. Identifying that WT SIX2+CITED1+ cells arise early in nephrogenesis and have an expression pattern favoring proliferation over differentiation that overlaps considerably with the expression pattern of both the tumor of origin and with the expression pattern of WT they generate after grafting.

Project description:Purpose: Bulk transcriptomics analysis of Wilms tumor SIX2+CITED1+ cells to compare and identify unique nephron progenitor transcriptome profiling (RNA-seq) signature between unfavorable and favorable Wilms Tumors and against human fetal kidney Methods: Wilms tumor samples were collected and transported on ice at 4°C in RPMI-1640 and a single cell suspensions were prepared following mechanical and enzymatic dissociation as previously publication. Enzymatic dissociation was performed with 125 U/ml collagenase I in RPMI-1640 at 37 °C for 35 min. The digested cells were then passed through a 100-μm cell strainer and a 40-μm cell strainer with washes of 1x PBS. The cell suspension was than centrifuged at 1500 rpm for 5 min and erythrocytes were eliminated using a red blood cell lysis kit. WT SIX2+CITED1+ cells were isolated using SIX2-Cy5 and CITED1-Cy3 Smartflare RNA probes following manufacturer’s instructions and previous publication. Briefly, cells were incubated over night at 37 °C with both RNA probes diluted at 1:20 in PBS and 25ul/ml in RPMI-1640 supplemented with 5% FBS, and 0.2% antimicrobial agent Primocin. After 16-18 h, cells were dissociation using TrypLE 1x for 5 min, cells were centrifuged at 1500 rpm for 5 min and prepared for FACS. RNA extraction was performed immediately after FACS using the RNeasy Micro Kit following manufacturer’s recommendations. After cDNA production and construction of DNA libraries, the samples were run on an Illumina NextSep500 (Illumina). Differential gene expression was analyzed using ERCC ExFold probes with the Remove Unwanted Variation R/Bioconductor software package combined with edgeR Results: Transcriptomics analysis of Wilms Tumor SIX2+CITED1+ cells in comparison to different tumor types and human fetal kidneys confirmed the nephrogenic signature of hFK-SIX2+CITED1+ and WT-SIX2+CITED1+ cells but highlighted differences in expression of pluripotency and self renewal-related genes like OCT4, FOXO1, SALL, NANOG along with a lower expression of β-catenin, TCF, and other growth factors known to promote differentiation (BMPs, FGFs). Hierarchical clustering of gene expression showed shared similarities between Wilms tumor samples against human fetal kidney samples however, with major differences in gene expression between tumor-to-tumor type was also present. Conclusion: Our study represents the first transcriptomic characterization of Wilms Tumor cancer stem cells (SIX2+CITED1+) against human fetal kidney SIX2+CITED1+ cells. Identifying specify gene expression and signaling pathway profiles across different subtypes of Wilms Tumor and against human fetal kidney SIX2+CITED1+ cells.

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Single cell transcriptomics of fetal kidney and Wilms tumor nephrogenic progenitors

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