Direct Isolation and Characterization of Human Nephron Progenitors.
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE74450 | GEO | 2016/09/16
SECONDARY ACCESSION(S): PRJNA300441
REPOSITORIES: GEO
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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.
2023-04-30 | GSE175698 | GEO
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.
2024-01-08 | GSE176342 | GEO
Project description:Human pluripotent stem cells (hPSC) provide a possible source for generation of kidney cells and organoids applicable in regenerative medicine, disease modeling and drug screening. By analyzing the effect of different factors on renal differentiation, we established an 8-day-protocol that steered hPSCs to the renal lineage by a step-wise process, segmented into mesoderm induction, intermediate mesoderm specification and metanephric induction outlining renal organogenesis. The differentiated cells contain populations of SIX2+/CITED1+ metanephric mesenchyme- (MM) and of HOXB7+/GRHL2+ ureteric bud (UB)-like cells at the end of 6 days. Transcriptome analysis corroborated that the in vitro generated precursor cell types at day 8 resemble their renal vesicle counterparts in vivo. The cells were further differentiated in 2-dimensional culture into podocyte- and diverse tubular epithelial-like cell types, showing their nephrogenic potential. In 3-dimensional culture, the progenitors gave rise to renal vesicles, and upon mouse embryonic kidney re-aggregation they form tubular structures.
2018-11-08 | GSE75711 | GEO
Project description:In developing mammalian kidney, nephron progenitor cells (NPC) give rise to all cells in mature nephrons. Expression of the transcription factor Six2 marks NPC in developing mouse kidneys. Within the Six2+ cell population, uncommitted NPC is marked by Cited1 expression. Towards the depletion of NPC in P2, most of the Cited1+ NPC is committed. Therefore, most of the Six2+ cells in kidney represents committed NPC. In order to explore the mechanism of NPC self-renewal and differentiation, hereby we generated transcriptionl profiles of uncommitted NPC (Cited1RFP+ cells from kidneys of E16.5 Cited1tagRFP transgenic mice) and committed NPC (Six2GFP+ cells from kidneys of P2 Six2TGC transgenic mice).
2018-01-18 | GSE89264 | GEO
Project description:Transcriptional profiling of zebrafish nephrogenic stem cells (lhx1a+) vs. zebrafish renal tubular cells (cdh17+): 2 color design, 3 biological replicates Two-condition experiment, renal tubule vs. nephrogenic stem cells. 3 biological replicates
2011-11-01 | E-GEOD-24803 | biostudies-arrayexpress
Project description:Recent studies using human pluripotent stem cells (hPSCs) have developed protocols to induce kidney-lineage cells and reconstruct kidney organoids. However, the separate generation of metanephric nephron progenitors (NPs), mesonephric NPs and ureteric bud (UB) cells, which constitute embryonic kidneys, in in vitro differentiation culture systems has not been fully investigated. Here we created a culture system in which these mesoderm-like cell types as well as paraxial and lateral plate mesoderm-like cells were separately generated from hPSCs. We succeeded in recapitulating nephrogenic niches from separately induced metanephric NP-like and UB-like cells, which were subsequently differentiated into glomeruli, renal tubules and collecting ducts in vitro and further vascularized in vivo. Our selective differentiation protocols should contribute to understanding the mechanisms underlying human kidney development and diseases and also supply cell sources for regenerative therapies.
2020-07-15 | GSE146118 | GEO
Project description:Recent studies using human pluripotent stem cells (hPSCs) have developed protocols to induce kidney-lineage cells and reconstruct kidney organoids. However, the separate generation of metanephric nephron progenitors (NPs), mesonephric NPs and ureteric bud (UB) cells, which constitute embryonic kidneys, in in vitro differentiation culture systems has not been fully investigated. Here we created a culture system in which these mesoderm-like cell types as well as paraxial and lateral plate mesoderm-like cells were separately generated from hPSCs. We succeeded in recapitulating nephrogenic niches from separately induced metanephric NP-like and UB-like cells, which were subsequently differentiated into glomeruli, renal tubules and collecting ducts in vitro and further vascularized in vivo. Our selective differentiation protocols should contribute to understanding the mechanisms underlying human kidney development and diseases and also supply cell sources for regenerative therapies.
2020-07-15 | GSE146117 | GEO
Project description:All nephrons in the mammalian kidney arise from a transient nephron progenitor population that is lost after birth. The recreation of nephron progenitors (NPs) and/or their maintenance in culture has been a major focus of renal regenerative strategies. Using a lentiviral screening approach, we previously generated an induced nephron progenitor (iNP) state in vitro using a pool of six transcription factors (SIX1, SIX2, HOXA11, OSR1, EYA1, SNAI2). Here, using an inducible piggyBac transposon system, we demonstrate efficient reprogramming to iNPs with only three transcription factors (SNAI2, EYA1 and SIX1). Coupled with maintenance in culture conditions supportive of this progenitor state, the resulting population can contribute to developing nephrons in vitro, ex vivo and in vivo. This approach provides a rapid and efficient method for the generation of NPs from human somatic cells.
2019-11-28 | GSE107410 | GEO
Project description:Nephron endowment is a key determinant of later life hypertension and kidney disease. Here we studied whether epigenetic changes, specifically the ten–eleven translocation (Tet) DNA demethylase family, Tet1, Tet2, and Tet3-mediated active DNA hydroxymethylation is necessary for gene expression regulation and kidney differentiation. We generated mice with deletion of Tet1, Tet2 or Tet3 in Six2 positive nephron progenitors (NP). We did not observe changes in development or kidney function in mice with nephron progenitor-specific deletion of Tet1, Tet2, Tet3 or Tet1/Tet2 or Tet1/Tet3. On the other hand, mice with combined Tet2 and Tet3 loss in Six2-positive NPCs failed to form nephrons leading to kidney failure and perinatal death. Tet2 and Tet3 loss in Six2-positive NPs resulted in defect in mesenchymal to epithelial transition and renal vesicle differentiation. Whole genome bisulfite sequencing, single cell RNA sequencing, and gene and protein expression assay identified a defect in expression in genes in the WNT-β-catenin signaling pathway in absence of Tet2 and Tet3 due to a failure in demethylation of these loci. Our results indicate the key role of Tet2 and Tet3-mediated active cytosine hydroxymethylation in NPs in kidney development and nephron endowment.
2022-11-23 | GSE212715 | GEO
Project description:Nephron endowment is determined by the self-renewal and induction of a nephron progenitor pool established at the onset of kidney development. In the mouse, the related transcriptional regulators Six1 and Six2 play non-overlapping roles in nephron progenitors. Transient Six1 activity prefigures, and is essential for, active nephrogenesis. In contrast, Six2 maintains later progenitor self-renewal from the onset of nephrogenesis. We compared Six2’s regulatory actions in mouse and human nephron progenitors by chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq). Surprisingly, SIX1 was identified as a SIX2 target unique to the human nephron progenitors. Further, RNA-seq and immunostaining revealed overlapping SIX1 and SIX2 progenitor activity in the 16 week human fetal kidney. Human SIX1 ChIP-seq revealed a similar set of targets to SIX2, and predicted both factors bind DNA through an identical recognition site. In contrast to the mouse where Six2 binds its own enhancers but doesn’t interact with DNA around Six1, both human SIX1 and SIX2 bind homologous SIX2 enhancers and putative enhancers positioned around SIX1. Transgenic analysis of a putative human SIX1 enhancer in the mouse revealed a transient, mouse-like, pre-nephrogenic, Six1 regulatory pattern. Together, these data demonstrate a divergence in SIX-factor regulation between mouse and human nephron progenitors. In the human, an auto/cross-regulatory loop drives continued SIX1 and SIX2 expression during active nephrogenesis. In contrast, the mouse establishes only an auto-regulatory Six2 loop. It is tempting to speculate that differential SIX-factor regulation may contribute to species differences in the duration of progenitor programs and nephron output.
2016-01-31 | GSE73865 | GEO