Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse aorta-gonad-mesonephros(AGM) derived stromal cells

ABSTRACT: A mouse AGM-derived cell line, AGM-s3, was shown to support the development of hematopoietic stem cells. To elucidate the molecular mechanisms regulating early hematopoiesis, we obtained subclones from AGM-s3, some of which were hematopoiesis supportive (s3-A9) and others which were non-supportive (s3-A7), and we analyzed the gene expression profiles by gene chip analysis. Experiment Overall Design: Genome-wide gene expression was examined using Affymetrix GeneChip array. Assays were performed according to the manufacturer's protocol. Total RNA was isolated from each stromal cell lines. We analysed 3 cell lines, AGM-s3-A9, AGM-s3-A7 and OP9. AGM-s3-A9 and OP9 are hematopoiesis supportive cell lines. AGM-s3-A7 is a hematopoiesis non-supportive cell line.

ORGANISM(S): Mus musculus

SUBMITTER: Kenji Nagao

PROVIDER: E-GEOD-11891 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Blood flow promotes emergence of definitive hematopoietic stem cells (HSCs) in the developing embryo, yet the signals generated by hemodynamic forces that influence hematopoietic potential remain poorly defined. In transplantation assays of hematopoietic reconstitution, we find that fluid shear stress endows long-term multilineage engraftment potential upon early hematopoietic tissues at E9.5 not previously described to harbor HSCs. Effects on hematopoiesis appear to be mediated in part by prostaglandin E2 (PGE2) and the cyclic AMP-protein kinase A (cAMP-PKA) signaling axis. Studies of Ncx1 cardiac mutants corroborate that blood flow is required for sufficient COX2 levels and phosphorylation of CREB. Further implicating PGE2 in mediating the effects of shear stress, we find that E10.5 and E11.5 AGM treated transiently with the synthetic analog dmPGE2 engraft more robustly and contribute to greater lymphoid reconstitution. These data provide a mechanism by which biomechanical forces induced by blood flow modulate hematopoietic potential. - AGM from C57BL/6J embryos at 10.5 days gestation (E10.5) were isolated by microdissection from uteri of pregnant dams, following by gentle dissociation by Accutase with agitation at room temperature for 20 minutes. Single cell suspension was seeded on microfluidic IBIDI VI^0.4 6-channel slides (0.8 to 1x10^7 cells per channel) and permitted to attach for 8 hours. Fluid movement was then applied to each channel using a Harvard Apparatus PHD ULTRA programmable syringe pump for manangement of M5300 Myelocult medium. Cells were exposed either to static/low flow (0.0001 dyne/cm^2) or wall shear stress (WSS) of 5 dyne/cm^2 for 6 hours or 36 hours. In addition, some cells were treated with 10 uM indomethacin (indo) to inhibit COX2 activity and PGE2 synthesis. Upon collection of cells with RLT lysis buffer (QIAGEN RNeasy kit), six channels of identical treatment were pooled to comprise a single sample. 24 samples total are included in this study. 12 samples were collected after 6 hours and 12 after 36 hours. In detail, samples included at 6 hours: 3 static, 3 static with indo, 3 WSS, 3 WSS with indo; and at 36 hours: 3 static, 3 static with indo, 3 WSS, 3 WSS with indo. Sample labels begin with the timepoint collected and end with the replicate number, i.e., 06WSS1 for the 6 hour collection of the first replicate of the WSS sample.

Project description:Hematopoietic stem cells (HSCs) are at the basis of the hematopoietic hierarchy. Their ability to self-renew and differentiate is strictly controlled by molecular signals produced by their surrounding micorenvironments composed of stromal cells. HSCs first emerge in the AGM (Aorta Gonads Mesonephros) region, amplify in the fetal liver (FL) and are maintained in the adult bone marrow (BM). To further characterize the molecular program of the HSC niches, we have compared the global transcriptome of HSC-supportive and non-supportive stromal clones established from the AGM, FL and BM. Hematopoietic stem cells (HSCs) are at the basis of the hematopoietic hierarchy. Their ability to self-renew and differentiate is strictly controlled by molecular signals produced by their surrounding micorenvironments composed of stromal cells. HSCs first emerge in the AGM (Aorta Gonads Mesonephros) region, amplify in the fetal liver (FL) and are maintained in the adult bone marrow (BM). To further characterize the molecular program of the HSC niches, we have compared the global transcriptome of HSC-supportive line from Fetal Calvaria (OP9) and non-supportive stromal clones from fetal liver (BFC). Hematopoietic stem cells (HSCs) are at the basis of the hematopoietic hierarchy. Their ability to self-renew and differentiate is strictly controlled by molecular signals produced by their surrounding micorenvironments composed of stromal cells. HSCs first emerge in the AGM (Aorta Gonads Mesonephros) region, amplify in the fetal liver (FL) and are maintained in the adult bone marrow (BM). To further characterize the molecular program of the HSC niches, we have compared the global transcriptome of HSC-supportive and non-supportive stromal clones established from fetal liver. We took advantage of stromal clones established from the AGM, FL and BM and tested for their ability to support or not HSCs ex vivo. RNA were extracted from confluent stromal cultures or sorted cells and used for hybridization of Affymetrix (mouse gene 1.0 ST) microarrays.

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Transcription profiling of mouse aorta-gonad-mesonephros(AGM) derived stromal cells

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