Aberrant expression of Podocalyxin (PODXL), a CD34 orthologue, has been associated with acute myeloid leukemia (AML). Herein, via tissue microarray, we discovered elevated PODXL expression in M2, M4 and M1 FAB-subtype patients. Importantly, various investigations have linked aberrant miRNA expression with AML (1). A miRNA prediction algorithm identified PODXL as a conserved target for miR-199b, a significantly down-regulated miRNA in AML. Further prediction of miR-199b-5p targets identified Discoidin domain receptor 1 (DDR1) as another highly conserved target. For the first time, IHC analyses showed that DDR1 levels were also highly up-regulated in AML and more significantly, were elevated in the same AML cases where PODXL levels were increased. Experimental validation (via-mimics) confirmed that both PODXL and DDR1 are targets of miR-199b-5p. Furthermore, 3’UTR-luciferase assays established that miR-199b-5p targets PODXL and DDR1. Most importantly, we found significant decrease in miR-199b-5p levels in most AML patients with elevated PODXL and DDR1 expressions. Importantly, overexpression of miR-199b-5p in K562 cells caused significant decrease in collagen IV induced migration. Taken together, our studies have identified concurrent increased expression of PODXL and DDR1 in AML and directly connect decreased miR-199b-5p to these novel targets and potential antigomir-mediated therapeutic implications in AML.
Human induced pluripotent stem cells (iPSCs) are a powerful tool for studying development and disease. However, different iPSC lines show considerable phenotypic variation. The lack of common well-characterized cell lines that are used widely frustrates efforts to integrate data across research groups or replicate key findings. Inspired by model organism communities who addressed this issue by establishing a limited number of widely accepted strains, we characterised candidate iPSC lines in unprecedented detail to select a well-performing line to underpin collaborative studies. Specifically, we characterised the morphology, growth rates, and single-cell transcriptomes of iPSC lines in the pluripotent state and assessed their genomic integrity using karyotyping, DNA microarrays, whole genome sequencing, and functional assays for p53 activity. We further tested their ability to be edited by CRISPR/Cas9 and used single-cell RNA sequencing to compare the efficiency with which they could be differentiated into multiple lineages. We found that there was significant variability in the performance of lines across the tested assays that enabled the rational selection of a lead line, KOLF2.1J, which is a gene-corrected derivative of a publicly available line from the Human Induced Pluripotent Stem Cells Initiative (HipSci) resource. We are now using this line in an initiative from the NIH Center for Alzheimer’s and Related Dementias to derive hundreds of gene-edited and functionalized sub-clones to be distributed widely throughout the research community along with associated datasets, with the aim of promoting the standardisation required for large-scale collaborative science in the stem cell field.SummaryThe authors of this collaborative science study describe a deep characterization of widely available induced pluripotent stem cell (iPSC) lines to rationally select a line that performs well in multiple experimental approaches. Analysis of transcriptional patterns in the pluripotent state, whole genome sequencing, genomic stability after highly efficient CRISPR-mediated gene editing, integrity of the p53 pathway, and differentiation efficiency towards multiple lineages identified KOLF2.1J as a well-performing cell line. The widespread distribution and use of this line makes it an attractive cell line for comparative and collaborative efforts in the stem cell field.HighlightsDeep genotyping and phenotyping reveals KOLF2.1J as well-performing cell line that is readily distributed and could serve as common reference lineDespite rare copy-neutral loss of heterozygosity (CN-LOH) events, iPSC lines retain genomic fidelity after CRISPR/Cas9-based gene editingOur multifactorial pipeline serves as a blueprint for future efforts to identify other lead iPSC linesGraphical abstract
3520 IL-3, GM-CSF and G-CSF are predominant regulators for growth and differentiation of myeloid progenitors. Interestingly, they all signal via a common JAK2-STAT5 pathway in myeloid progenitor compartments. However, the specific mechanism through which JAK2-STAT5 responds differentially to early-acting and lineage restricted cytokines, particularly in leukemic and stem/progenitor cells, is largely unresolved. Aberrations in IL-3, GM-CSF and G-CSF induced signaling are frequently reported in acute myeloid leukemia (AML). microRNA (miRNA) play several crucial roles during hematopoiesis that include lineage decisions, stem cell progenitor transitions, niche control and other cell functions. Recent investigations have linked aberrant miRNA expression with AML. We hypothesized that a unique response of leukemic myeloid progenitors to IL-3, GM-CSF, and G-CSF are possibly mediated in part by distinct regulation at the miRNA level. Therefore, herein, by utilizing a unique leukemic myeloid cell line, AML-193, that responds to both early and late acting cytokines, we profiled IL-3, GM-CSF and G-CSF regulated miRNA signatures in leukemic myeloid progenitors. For miRNA profiling, AML-193 cells were initially exposed to IL-3 for 3 days followed by GM-CSF for 3 more days and subsequently to G-CSF for 3 days. We then profiled miRNA expression induced by IL-3, GM-CSF and G-CSF in AML-193 cells by treating the respective cohorts post growth factor deprivation with corresponding cytokines. Using SA Bioscience's complete Human V2.0 miRNA Genome Array platform for real-time qPCR-based miRNA expression profiling, we investigated miRNA signatures regulated by IL-3, GM-CSF and G-CSF for n=704 miRNAs in AML-193 cells. Frequencies of false positives were avoided using stringent filters. IL-3 specifically regulated 54 miRNAs and those miRNAs that were highly regulated included miR-362-3p, miR-590-3p, miR-340, miR-24-2, miR-1183 and miR-99a. GM-CSF specifically regulated miRNAs included let-7f, let-7a*, miR-195, miR-122, miR-376-c and miR-33a. G-CSF specifically regulated set included miR-21*, miR-192*, miR-32*, miR-7-1*, miR-545* and miR-37-4a*. Interestingly, 301 miRNAs were commonly regulated by IL-3, GM-CSF and G-CSF. Among the commonly regulated miRNAs, the ones that were subjected to high levels of regulation included miR-590-5p, miR-219-5p, miR-92-a1*, miR-378*, miR-548-3p, miR-29a*, miR-590-3p, miR-203, miR-363, miR-454, miR-340, miR-196a, miR-152, miR-10b, miR-24-2*, miR-10a, miR-182, miR-27a*, and miR-199a-3p. Interestingly, the commonly regulated miRNAs demonstrated a directional regulation in the order of IL-3>GM-CF>G-CSF. Analysis of the potential targets of significantly regulated miRNAs revealed important functional roles in myeloid cell development and differentiation. Conserved targets of miR-590-5p included BMPR2, PCBP2 and KLF3. Targets for miR-219-5p included Smad4 and GADD45b. Targets for miR-362-3p included Sox17, an inhibitor of the Wnt signaling pathway. Furthermore, to identify the JAK2-STAT5 responsiveness of miR-219-5p, miR-362-3p, and miR-590-5p, a STAT5 specific inhibitor (N′-((4-Oxo-4H-chromen-3-yl)methylene)nicotinohydrazide) was used prior to cytokine treatment. Interestingly, IL-3 and GM-CSF mediated induction of miR-590-5p, miR-219-5p and miR-362-3p were not significantly affected by STAT5 inhibitor. However, the G-CSF induction of miR-219-5p and miR-590-5p were significantly inhibited by the STAT5 specific inhibitor. In summary, we have discovered for the first time novel miRNA profiles regulated by IL-3, GM-CSF and G-CSF in an acute myeloid leukemia progenitor cell model. Importantly, we have established a cytokine regulated miRNome for leukemic myeloid progenitors and set the stage for future investigations in leukemic stem cells to delineate the pathological roles of dysregulated miRNAs in AML. In addition, our study displayed that G-CSF induced a subset of miRNAs that are principally regulated via the STAT5 circuit in AML. Further, these novel miRNA signatures may have therapeutic implications for targeting dysregulated miRNAs by antagomir strategy or miRNA replacement therapy, paving the way for the development of novel miRNA-based therapeutic interventions in AML. Disclosures: No relevant conflicts of interest to declare.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.