Surface Fucosylation Of CD44on Human Adipose-derived Stem Cells (ASCS) Augments Homing To Bone

Objective:1. To establish a method for isolating and cultivating hASCs derived from human adipose in vitro, in order to study their morphology, cell surface markers and biological properties.2. To establish a method for isolating and cultivating HUVEC, in order to study their morphology and biological properties.Methods:1. The hASCs were isolated from human adipose tissue with0.15%collagenase digesting,,;observed under inverted microscope and tested with CCK-8method, flow cytometry. Its differentiation potential was proved by osteogenic and adipogenic differentiation.2. The HUVEC were isolated from human umbilical cord with0.2%collagenase digesting, observed under inverted microscope and identified by the uptake of DiI-Ac-LDL.Results:1. After isolation and cultivation, we obtained a large amount fibroblast-like cells, which grow like swirls. The CCK-8results showed the cultivated cells have strong and stable proliferation activity. Flow cytometric analysis showed that mesenchymal stem cells markers CD29and CD44were positive; CD45and HLA-DR were negative. The cultivated cells could be functionally induced into adipocytes and osteoblasts in the presence of appropriate conditioned media.2. After isolation and cultivation, we obtained a large amount cobblestone-like cells. Under fluorescence microscope, the cultivated cells uptaked DiI-Ac-LDL.Conclusion:In order to provide cells for the follow-up experiments, we have developed efficient methods for isolation and cultivation of hASCs and HUVEC. Objective:Apply fucosylation in vitro to convert the inborn CD44glycoform on ASCs into hematopoietic cell E-selectin/L-selectin ligand (HCELL), which renders powerful E-selectin binding for functional studies and in vivo basis for the study.Methods:We used an a-l,3-fucosyltransferase under appropriate enzymatic conditions for fucosylation. First, we detected the expression of adhesion molecules on untreated hASCs via flow cytometic. Then, we used fucosylated hASCs to process functional study in vitro, including flow cytometic, western blotting, static adhesion assay and parallel plate flow chamber adhesion assay to evaluate the expression of sLex and E-selectin binding ability after fucosylation. Furthermore, we assessed the impact of exofucosylation on cell viability, proliferation, differentiation capacity.Results:Flow cytometric analysis demonstrated that CD29, CD44, CD49d, CD49e were positive while sLex, PSGL-1, CXCR4, β7were negative on untreated hASCs. And there is no binding between untreated hASCs and endothelial E-selectin. After fucosylation, flow cytometric analysis and western blotting showed that high expression of sLe and strong E-selectin binding. Compared to control, fucosylated hASCs showed powerful E-selectin binding under static or flow conditions. And there is no negative effect on cell viability, proliferation or multipotency after fucosylation.Conclusion:These data reveal that we convert the inborn CD44glycoform on ASCs into hematopoietic cell E-selectin/L-selectin ligand (HCELL) after fucosylation. The created HCELL renders hASCS powerful E-selectin binding ability under static or flow conditions. And ex vivo fucosylation has no harmful imapct on cell viability, proliferation or multipotency. Objective:Apply fucosylation in vitro to convert the CD44glycoform on hASCs into hematopoietic cell E-selectin/L-selectin ligand (HCELL), which has powerful E-selectin binding ability. And the enforced HCELL improves hASCs homing to bone in vivo.Methods:1. DiI labeled fucosylated hASCs were injected intravenously into NOD/SCID mice. Mice were killed16hours after injection, femora were removed, and marrow suspensions were assessed for frequencies of DiI-positive cells by flow cytometry.2. Fucosylated hASCs were injected intravenously into NOD/SCID mice. Mice were killed3months after injection, skull from each animal was harvested, fixed, decalcified and snap frozen in OCT. And5-μm frozen sections were obtained. Then, we performed immunofluorescent staining of bone frozen sections to determine the presence of human osteoid tissue.Results:1. Flow cytometric analysis demonstrated that fucosylated hASCs accumulated in the marrow more efficiently than untreat hASC, which indicated that the created HCELL on hASCs after fucosylation licenses immigration of these cells into the bone marrow.2. Sections of bone from mice that received untreated hASCs or HBSS alone completely lacked staining by mAb to human osteocalcin, whereas bone sections of all mice that received fucosylated hASCs showed sparse human osteocalcin postive cells localized to the endosteal surfaces. The result showed that the extravasated cells can lodge within marrow endosteal surfaces and generate human osteoid in mouse bone marrow.Conelusion:Enforced HCELL confers tropism to bone and ex vivo fucosylation might be a simple and effective method to enhance stem cell homing to bone marrows of patients receiving stem cell-based therapies.