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Maintenance of HSC by Wnt5a secreting AGM-derived stromal cell line.

ABSTRACT: The microenvironment wherein hematopoietic stem cells (HSC) reside orchestrates HSC self-renewal vs. differentiation decisions. Stromal cells derived from ontogenically divergent hematopoietic microenvironments can support HSC in vitro and have been used to decipher factors that influence HSC fate decisions. Employing stromal cell lines derived from the aorta-gonad-mesonephros and embryonic liver, we aim to identify secreted factors that maintain/expand HSC in vitro.We cultured murine lineage antigen-negative (Lin(-)) bone marrow cells in transwells above the UG26-1B6, urogenital ridge-, and EL08-1D2, embryonic liver-derived cell lines. We, also, performed real-time quantitative PCR analysis to identify differentially expressed genes from the Wnt family of proteins in ontogenically different stromal cell lines.Lin(-) murine bone marrow cells maintained for 3 weeks in transwells above UG26-1B6 but not EL08-1D2 cells contained competitive repopulating HSC. Addition of as few as 25% UG26-1B6 cells to EL08-1D2 feeders led to maintenance of HSC in noncontact cultures, validating soluble factors are secreted by the UG26-1B6 cells. As we found that Wnt5a was significantly higher expressed in UG26-1B6 than EL08-1D2 cells, we added Wnt5a to EL08-1D2 transwell cultures or an antibody against Wnt5a to UG26-1B6 transwell cultures. Addition of Wnt5a to EL08-1D2 transwell cultures restored maintenance of HSC, whereas addition of an anti-Wnt5a antibody to UG26-1B6 transwell cultures inhibited maintenance of competitive repopulating HSC.We demonstrate that stromal cell lines generated from embryonic microenvironments provide a tool to identify secreted proteins that play a role in the maintenance of HSC, and that at least one of the factors produced by UG26-1B6 cells responsible for preserving HSC is Wnt5a.

SUBMITTER: Buckley SM

PROVIDER: S-EPMC3031906 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Maintenance of HSC by Wnt5a secreting AGM-derived stromal cell line.

Buckley Shannon M SM Ulloa-Montoya Fernando F Abts David D Oostendorp Robert A J RA Dzierzak Elaine E Ekker Stephen C SC Verfaillie Catherine M CM

Experimental hematology 20101007 1

<h4>Objective</h4>The microenvironment wherein hematopoietic stem cells (HSC) reside orchestrates HSC self-renewal vs. differentiation decisions. Stromal cells derived from ontogenically divergent hematopoietic microenvironments can support HSC in vitro and have been used to decipher factors that influence HSC fate decisions. Employing stromal cell lines derived from the aorta-gonad-mesonephros and embryonic liver, we aim to identify secreted factors that maintain/expand HSC in vitro.<h4>Materia ...[more]

PMID: 20933051

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Project description:Human adipose-derived stromal vascular fraction (hSVF) cells are an easily accessible, heterogeneous cell system that can spontaneously self-assemble into functional microvasculatures in vivo. However, the mechanisms underlying vascular self-assembly and maturation are poorly understood, therefore we utilized an in vitro model to identify potential in vivo regulatory mechanisms. We utilized passage one (P1) hSVF because of the rapid UEA1+ endothelium (EC) loss at even P2 culture. We exposed hSVF cells to a battery of angiogenesis inhibitors and found that the pan-Wnt inhibitor IWP2 produced the most significant hSVF-EC networking decrease (~25%). To determine which Wnt isoform(s) and receptor(s) may be involved, hSVF was screened by PCR for isoforms associated with angiogenesis, with only WNT5A and its receptor, FZD4, being expressed for all time points observed. Immunocytochemistry confirmed Wnt5a protein expression by hSVF. To see if Wnt5a alone could restore IWP2-induced EC network inhibition, recombinant human Wnt5a (0-150 ng/ml) was added to IWP2-treated cultures. The addition of rhWnt5a significantly increased EC network area and significantly decreased the ratio of total EC network length to EC network area compared to untreated controls. To determine if Wnt5a mediates in vivo microvascular self-assembly, 3D hSVF constructs containing an IgG isotype control, anti-Wnt5a neutralizing antibody or rhWnt5a were implanted subcutaneously for 2w in immune compromised mice. Compared to IgG controls, anti-Wnt5a treatment significantly reduced vessel length density by ~41%, while rhWnt5a significantly increased vessel length density by ~62%. However, anti-Wnt5a or rhWnt5a did not significantly affect the density of segments and nodes, both of which measure vascular complexity. Taken together, this data demonstrates that endogenous Wnt5a produced by hSVF plays a regulatory role in microvascular self-assembly in vivo. These findings also suggest that manipulating Wnt signaling could enhance control of hSVF vascularization in tissue engineering applications.

Dataset Information

Maintenance of HSC by Wnt5a secreting AGM-derived stromal cell line.

Publications

Maintenance of HSC by Wnt5a secreting AGM-derived stromal cell line.

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