Project description:Recently the cancer stem cell (CSC) model has been put forward to describe how a subset of cells within the tumor is responsible for tumor growth and heterogeneity. Wilms' tumor (WT), the most common pediatric renal malignancy, arises from developmentally arrested early renal progenitors. WT NCAM1+ALDH1+ CSCs have been recently isolated and shown to localize to tumor blastema. Herein by generating 'blastema'-only WT xenografts composed solely by cells expressing the SIX2 and NCAM1 embryonic renal stem cell markers, we surprisingly show that sorted ALDH1+ WT CSCs are phenotypically not the earliest renal stem cells. Rather, gene expression and proteomic comparative analysis disclose a more differentiated self-renewing epithelial cell type than bulk of the blastema. Thus, WT CSCs do not represent the transformed counterpart of the most primitive renal stem cell being more differentiated than the presumable WT cell of origin and are likely to de-differentiate to propagate the tumor blastema. We used Wilms tumor Xns, as well as, fetal renal tissue originally obtained from a patients or aborted fetus
Project description:Recently the cancer stem cell (CSC) model has been put forward to describe how a subset of cells within the tumor is responsible for tumor growth and heterogeneity. Wilms' tumor (WT), the most common pediatric renal malignancy, arises from developmentally arrested early renal progenitors. WT NCAM1+ALDH1+ CSCs have been recently isolated and shown to localize to tumor blastema. Herein by generating 'blastema'-only WT xenografts composed solely by cells expressing the SIX2 and NCAM1 embryonic renal stem cell markers, we surprisingly show that sorted ALDH1+ WT CSCs are phenotypically not the earliest renal stem cells. Rather, gene expression and proteomic comparative analysis disclose a more differentiated self-renewing epithelial cell type than bulk of the blastema. Thus, WT CSCs do not represent the transformed counterpart of the most primitive renal stem cell being more differentiated than the presumable WT cell of origin and are likely to de-differentiate to propagate the tumor blastema.
Project description:When assembling a nephron during development a multipotent stem cell pool becomes restricted as differentiation ensues. A faulty differentiation arrest in this process leads to transformation and initiation of a Wilms' tumor. Mapping these transitions with respective surface markers affords accessibility to specific cell subpopulations. NCAM1 and CD133 have been previously suggested to mark human renal progenitor populations. Herein, using cell sorting, RNA sequencing, in vitro studies with serum-free media and in vivo xenotransplantation we demonstrate a sequential map that links human kidney development and tumorigenesis; In nephrogenesis, NCAM1+CD133- marks SIX2+ multipotent renal stem cells transiting to NCAM1+CD133+ differentiating segment-specific SIX2- epithelial progenitors and NCAM1-CD133+ differentiated nephron cells. In tumorigenesis, NCAM1+CD133- marks SIX2+ blastema that includes the ALDH1+ WT cancer stem/initiating cells, while NCAM1+CD133+ and NCAM1-CD133+ specifying early and late epithelial differentiation, are severely restricted in tumor initiation capacity and tumor self-renewal. Thus, negative selection for CD133 is required for defining NCAM1+ nephron stem cells in normal and malignant nephrogenesis. Human fetal kidney mRNA profiles of 3 cell populations (NCAM1+/CD133-, NCAM+/CD133+, NCAM-/CD133+) were generated by deep sequencing using Illumina HiSeq.
Project description:Wilms tumor, or nephroblastoma, is the most common pediatric kidney cancer, and is known to arise from renal precursor cells in the fetal kidney. Given that single nuclei RNA-sequencing generates gene expression profiles of diverse cell types within a heterogeneous tissue, here we sought to utilize this technology to profile blastema, stroma, and epithelial components of favorable histology Wilms tumor samples.
Project description:When assembling a nephron during development a multipotent stem cell pool becomes restricted as differentiation ensues. A faulty differentiation arrest in this process leads to transformation and initiation of a Wilms' tumor. Mapping these transitions with respective surface markers affords accessibility to specific cell subpopulations. NCAM1 and CD133 have been previously suggested to mark human renal progenitor populations. Herein, using cell sorting, RNA sequencing, in vitro studies with serum-free media and in vivo xenotransplantation we demonstrate a sequential map that links human kidney development and tumorigenesis; In nephrogenesis, NCAM1+CD133- marks SIX2+ multipotent renal stem cells transiting to NCAM1+CD133+ differentiating segment-specific SIX2- epithelial progenitors and NCAM1-CD133+ differentiated nephron cells. In tumorigenesis, NCAM1+CD133- marks SIX2+ blastema that includes the ALDH1+ WT cancer stem/initiating cells, while NCAM1+CD133+ and NCAM1-CD133+ specifying early and late epithelial differentiation, are severely restricted in tumor initiation capacity and tumor self-renewal. Thus, negative selection for CD133 is required for defining NCAM1+ nephron stem cells in normal and malignant nephrogenesis.
Project description:Mouse spermatogonial stem cells (SSCs) continuously self-renew on the feeder layers in serum-free culture medium supplemented with glial cell line-derived neurotrophic factor and fibroblast growth factor 2. To identify novel nuclear proteins involved in SSC maintenance, comparative proteomic profiling of nuclear proteins was performed between self-renewing and differentiation-initiated SSCs in culture. The self-renewing SSC cultures were established from C57BL/6 mouse testes. Nuclear fractions from self-renewing SSC cultures treated with ethanol as a vehicle control (spermatogonial stem cells) and differentiation-initiated SSC cultures treated with 0.3 μM retinoic acid for 24 h (spermatogonial progenitor cells) were isolated for proteomic analysis.
Project description:We performed single-cell RNA-seq of human iPSC-derived long-term self-renewing neural epithelial stem cells (hiPSC-lt-NES cells) using Quartz-seq methods to characterize cellular heterogeneity .
Project description:To identify cell-populations within human iPSC-derived long-term self-renewing neural epithelial stem cells (hiPSC-lt-NES cells) which retain capacities to generate undesired grafts, we performed gene expression microarray analysis.
Project description:To identify cell-populations within human iPSC-derived long-term self-renewing neural epithelial stem cells (hiPSC-lt-NES cells) which retain capacities to generate undesired grafts, we established single cell-derived hiPSC-lt-NES cell clones and performed gene expression microarray analysis.