TY - JOUR
T1 - Maintenance of HSC by Wnt5a secreting AGM-derived stromal cell line
AU - Buckley, Shannon M.
AU - Ulloa-Montoya, Fernando
AU - Abts, David
AU - Oostendorp, Robert A.J.
AU - Dzierzak, Elaine
AU - Ekker, Stephen C.
AU - Verfaillie, Catherine M.
N1 - Funding Information:
The authors wish to thank Vick Van Duppen for help with FACS sorting, Sarah Schouteden for technical assistance, and Dr. Rik Snoeckx for his critical review of all data for accuracy and the manuscript before submission. This work was supported by PO1-CA-65493-06 (C.M.V.), FWO G.0451.06 (C.M.V.), KUL CoE (C.M.V.), FWO-Odysseus Fund (C.M.V.), FP6-STREP CHRYSTAL (C.M.V.), NIH RO1 DK0504077 (E.D.), and DFG-008/2 (R.O.).
PY - 2011/1
Y1 - 2011/1
N2 - Objective: 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. Materials and Methods: 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. Results: 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. Conclusions: 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.
AB - Objective: 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. Materials and Methods: 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. Results: 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. Conclusions: 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.
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U2 - 10.1016/j.exphem.2010.09.010
DO - 10.1016/j.exphem.2010.09.010
M3 - Article
C2 - 20933051
AN - SCOPUS:78650205344
SN - 0301-472X
VL - 39
SP - 114-123.e5
JO - Experimental Hematology
JF - Experimental Hematology
IS - 1
ER -