Method for differentiation of stem cells into vascular cells and the induction of angiogenesis using the same
Inventors
Kim, Sang-Heon • Kim, Soo Hyun • Park, In Su • Jung, Young Mee
Assignees
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Publication Number
US-9644183-B2
Publication Date
2017-05-09
Expiration Date
Abstract
A method for differentiating stem cells into vascular cells, including adhering the stem cells to a culture plate with a surface having a hydrophobic property or on which a growth factor is immobilized, and culturing the cells. The cultured stem cells later detach from the culture plate as their density increases to form a three-dimensional cell cluster and differentiate into vascular cells. The cell cluster can be used as a cell therapy agent for angiogenesis.
Core Innovation
The invention relates to differentiating adipose stem cells into vascular endothelial cell clusters for the induction of angiogenesis. Adipose stem cells are applied to a culture plate having a hydrophobic surface and a growth factor immobilized thereon, where the growth factor is fibroblast growth factor (FGF), and the stem cells adhere to the surface by physical attraction to the hydrophobic surface and by biological attraction to the growth factor.
The stem cells are cultured while attached to the surface, and a three-dimensional cell cluster is formed and detached from the culture plate so as to float in the culture medium. The three-dimensional cell cluster is further cultured in the culture medium to induce differentiation of the stem cells into vascular endothelial cells.
The resulting cell cluster is associated with angiogenic behavior and expression of vascular endothelial-associated markers. The three-dimensional clusters are reported to lead to a hypoxia-related intracellular response, with subsequent upregulation of angiogenesis-related stimulators such as VEGF, angiogenin, and IL-8.
Claims Coverage
The document includes one independent claim directed to a method that differentiates adipose stem cells into vascular endothelial cell clusters for induction of angiogenesis, comprising immobilized FGF on a hydrophobic surface, formation of floating three-dimensional clusters, and subsequent culturing to induce differentiation. Dependent claims refine the hydrophobic surface materials, growth factor immobilization configuration using a polypeptide linker recombinant fusion, and a cluster diameter constraint.
Hydrophobic FGF-immobilized culture plate for initial adhesion and attachment
A method comprising applying adipose stem cells to a culture plate having a hydrophobic surface and a growth factor immobilized thereon, wherein the growth factor is fibroblast growth factor (FGF), and allowing the stem cells to adhere to the surface by physical attraction to the hydrophobic surface and by biological attraction to the growth factor.
Floating three-dimensional cluster formation after detachment from the plate
Culturing the stem cells in a culture medium while attached to the surface and subsequently forming a three-dimensional cell cluster detached from the culture plate and floating in the culture medium.
Post-formation three-dimensional culturing to induce vascular endothelial differentiation
Culturing the three-dimensional cell cluster in the culture medium to induce differentiation of the stem cells into vascular endothelial cells.
Selected hydrophobic surface polymer group
The hydrophobic surface polymer is selected from polystyrene, polymethylmethacrylate (PMMA), polyethylene terephthalate (PET), polyvinylchloride (PVC), polyethylene (PE), polypropylene (PP), polytetrafluoroethylene (PTFE), poly(L-lactic acid) (PLLA), poly(D,L-lactic acid) (PDLLA), polyglycolic acid (PGA), poly(caprolactone) (PCL), poly(hydroxyalkanoate), polydioxanone (PDS), polytrimethylencarbonate, and copolymers.
Selected hydrophobic surface metal group
The hydrophobic surface metal is selected from stainless steel, titanium, gold, or platinum.
Polypeptide-linker FGF recombinant fusion for growth factor immobilization
FGF is immobilized using a polypeptide linker-growth factor recombinant protein in which the amino terminus of fibroblast growth factor (FGF) is fused to the carboxyl terminus of the polypeptide linker, where the polypeptide linker-growth factor recombinant protein has the amino acid sequence of SEQ ID NO: 1.
Cluster diameter constraint
The three-dimensional cell cluster has a diameter ranging from 400 µm to 1 mm.
Across the independent and refined features, the method centers on culturing adipose stem cells on an FGF-immobilized hydrophobic culture plate, forming floating three-dimensional clusters by detachment, and then culturing those clusters to induce differentiation into vascular endothelial cells, with further specifications for surface materials, growth factor immobilization via a polypeptide-linker recombinant fusion, and a cluster diameter range.
Stated Advantages
In vivo transplantation yields many blood-vessel or tubular structures with positive staining for human vascular markers.
In an ischemic rat hindlimb model, the cell cluster increases blood flow and angiogenic marker staining versus controls.
Documented Applications
In vivo transplantation of the differentiated cell cluster in nude mice, including formation of blood-vessel/tubular structures with positive staining for human vascular markers.
Use in an ischemic rat hindlimb model to increase blood flow and angiogenic marker staining versus controls.
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