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Series GSE114464 Query DataSets for GSE114464
Status Public on Apr 26, 2022
Title Mesoderm-derived PDGFRA+ cells regulate emergence of hematopoietic stem cells in the dorsal aorta
Organism Mus musculus
Experiment type Expression profiling by high throughput sequencing
Summary During embryonic development, the first hematopoietic stem cells (HSCs) arise from a transient population of endothelial cells lining the ventral surface of the dorsal aorta, via a process of endothelial to hematopoietic transition (EHT) at embryonic day (E) 10.5. This region contains resident PDGFRA+ stromal cells (PSCs), but their identity and role in HSC generation in the AGM are not well understood. Using a library of compound transgenic mice, we identified a population of PDGFRA+/Nestin-GFP (N-GFP)-/PDGFRB-/CD31- cells with PSC activity in the E10.5 and E11.5 mouse AGM. Freshly isolated PSCs were adept at forming blood vessels with CD31+ luminal endothelium enveloped by PDGFRB+ pericytes, when transplanted subcutaneously into mice. Conditional ablation of PDGFRA+ or Nestin+ cells led to either complete or partial loss of PSCs respectively, with severe loss of endothelial and pericyte-like cells, and concomitant loss of blood formation in the AGM. Lineage tracing studies using tamoxifen induction in PDGFRACreERT2/R26eYFP embryos showed that stromal, sub-endothelial, endothelial and long-term repopulating hematopoietic stem cells (LT-HSCs) cells in the E11.5 AGM were progeny of PDGFRA cells. Using transgenic reporter mice, we showed that MesP1 (mesoderm) derived PDGFRA+ cells dominated the sub-endothelial and deeper ventral stroma in the AGM at E10.5 and E11.5 but were replaced by Wnt1 (neural crest) derived cells at E13.5. Re-aggregation of E11.5 Mesp1 derived PSC cells with E13.5 aortic or adult cardiac non-hemogenic endothelial cells resulted in the generation of endothelial cell derived LT-HSCs. RNA-sequencing analysis of non-hemogenic E13.5 endothelial cells showed up-regulation of EHT genes, WNT, BMP and Notch cell signalling pathways when re-aggregated with E11.5 Mesp1 derived PSCs. LT-HSC generation from these re-aggregates was suppressed by dose-dependent inhibition of PDGF-AA/PDGFRA signalling. Taken together, we report that PSC populations in the AGM are temporally dynamic, and that MesP1-derived PSCs regulate hemogenic potential of the endothelium and that this cooperativity is dependent on PDGF-AA/PDGFRA signalling.
This submission represents the RNAseq component of the study.
Overall design E11.5- and E13.5 AGM PSCs were isolated from Mesp1DsRed and Wnt1DsRed embryos and cultured as previously described. Dissected AGMs were transferred into collagenase Type II (263 U/ml; Worthington Biosciences, Lakewood, NJ) and placed on a shaker at 37˚C for 15 min. The supernatant was passed through a 40 µm filter into a fresh tube and inactivated with 100% FCS. Cells were washed twice in 2% FCS in PBS and plated in alpha-MEM (Invitrogen, Carlsbad, CA) with 20% FCS, and penicillin/ streptomycin/ glutamine (P/S/G, Invitrogen)2 and cultured in the incubator at 37˚C, 5% CO2 for 72 h. At the end of 72 hrs, cells were washed in PBS to remove non-adherent cells and cultures were continued in fresh medium. Cells were passaged on reaching 80% confluence. After passaging, cells were placed back in tissue culture flasks with alpha-MEM + 20% FCS + P/S/G for bulk passaging. Cells were routinely cryopreserved in 10% DMSO and 90% culture medium. Re-aggregates were made by reconstituting FAC sorted cells from one AGM equivalent or 50,000 adult cardiac endothelial cells with 200,000 AGM PSCs. Dissociated cells were resuspended in 10μl of IMDM+ (IMDM Invitrogen) containing 20% fetal calf serum, 4mM L-glutamine, 50units/ml penicillin/streptomycin, 0.1mM mercaptoethanol, 100ng/ml IL, 100ng/ml SCF and 100ng/ml Flt3L (Peprotech). Re-aggregates were made by centrifugation in a yellow tip occluded by parafilm at 300 x g for 5 min and cultured on top of a 0.65μm Durapore filter (Millipore, Cat. No. DVPP02500) at the gas-liquid interface as described. Tissues were maintained in 5% CO2 at 370C in a humidified incubator.
Contributor(s) Chandrankanthan V, Huang Y, Beck D
Citation(s) 35902769
Submission date May 15, 2018
Last update date Mar 23, 2023
Contact name Yizhou Huang
Organization name University of New South Wales
Street address High Street
City UNSW Sydney
State/province NSW
ZIP/Postal code 2052
Country Australia
Platforms (1)
GPL21273 HiSeq X Ten (Mus musculus)
Samples (27)
GSM3142249 E11.Mesp1+A+ PSC rep1
GSM3142250 E11.Mesp1+A+ PSC rep2
GSM3142251 E11.Mesp1+A+ PSC rep3
BioProject PRJNA471497
SRA SRP146063

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Supplementary file Size Download File type/resource
GSE114464_RAW.tar 2.2 Gb (http)(custom) TAR (of BW)
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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