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DDX1-Mediated mitochondrial DNA G-quadruplex Resolution Is Essential for Hematopoietic Stem Cell Homeostasis and Radioresistance

ABSTRACT: Sorted CD34+ hematopoietic stem cells (HSCs) were cultured overnight at 37°C in a humidified incubator with 5% CO₂. The cells were then exposed to 5 Gy X-ray irradiation and cultured for an additional 24 hours. For each experimental condition (irradiated and non-irradiated), 1*10^7 CD34+ HSCs were collected. Mitochondrial proteins were extracted using the Mitochondrial Protein Extraction Kit (Beyotime, Cat. No. C3601). Subsequent LC-MS/MS analysis, sequence database searching, and bioinformatics analyses were conducted by Bioprofile Co., Ltd.

ORGANISM(S): Homo Sapiens

SUBMITTER: jiankun Fan

PROVIDER: PXD058114 | iProX | Thu Nov 21 00:00:00 GMT 2024

REPOSITORIES: iProX

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Project description:The cellular ontogeny of hematopoietic stem cells (HSCs) remains poorly understood because their isolation from and their identification in early developing small embryos are difficult. We attempted to dissect early developmental stages of HSCs using an in vitro mouse embryonic stem cell (ESC) differentiation system combined with inducible HOXB4 expression. Here we report the identification of pre-HSCs and an embryonic type of HSCs (embryonic HSCs) as intermediate cells between ESCs and HSCs. Both pre-HSCs and embryonic HSCs were isolated by their c-Kit+CD41+CD45- phenotype. Pre-HSCs did not engraft in irradiated adult mice. After co-culture with OP9 stromal cells and conditional expression of HOXB4, pre-HSCs gave rise to embryonic HSCs capable of engraftment and long-term reconstitution in irradiated adult mice. Blast colony assays revealed that most hemangioblast activity was detected apart from the pre-HSC population, implying the early divergence of pre-HSCs from hemangioblasts. Gene expression profiling suggests that a particular set of transcripts closely associated with adult HSCs is involved in the transition of pre-HSC to embryonic HSCs. Experimental strategy consisted of EB formation, co-culture with OP9 cells, and functional assays. iHOXB4 ESCs were allowed to differentiate spontaneously into EBs for 6 days without HOXB4 expression. We decided to fractionate EB6 cells mainly because by day 6 of culture the number of multipotent progenitors reaches a plateau and several surface markers become detectable. CD41 is known as a marker for the initiation of definitive hematopoiesis. CD41 appeared in a significant proportion of EB cells on day 6 of culture. Induced HOXB4 expression during EB formation did not affect the generation of colony forming cells and repopulating cells in the OP9 and iHOXB4 system or the appearance of surface markers in EB cells. EB6 cells were analyzed and sorted by flow cytometry. Sorted EB6 cells were co-cultured with OP9 cells for various days under HOXB4-on or -off conditions. The minimal requirement of the co-culture period appeared to be only 4 days, which is much shorter than previously thought. After a second analysis and fractionation by flow cytometry, cells were subjected to in vivo repopulating assays under HOXB4-on or -off conditions.

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DDX1-Mediated mitochondrial DNA G-quadruplex Resolution Is Essential for Hematopoietic Stem Cell Homeostasis and Radioresistance

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