Behavior of multipotent stem cells isolated in mobilized peripheral blood from sheep after culture with human chondrogenic medium

摘要

Today, regenerative medicine requires new sources of multipotent stem cells for their differentiation to chondrocytes using the mediums of differentiation available in the market. This study aimed to determine whether the Mesenchymal Stem Cells (MSCs) isolated from Mobilized Peripheral Blood (MPB) in sheep using the Granulocyte Colony-Stimulating Factor (G-CSF), have the ability of first acquire a fibroblast-like morphology after being forced out of the bone marrow niche by G-CSF and second, if the cells have the capacity to express collagen type-II alpha I in primary culture using a human commercial media of differentiation. Six Suffolk male sheep with age of 2 years were mobilized using G-CSF. One subcutaneous injection of 10 mcg per kilogram of bodyweight were administered every 24 h during three consecutive days. At day four, a sample of 20 mL of peripheral blood was harvested, afterwards, monocytes cells were separated by ficoll gradient. The mobilized MSCs were expanded in primary culture in DMEM medium supplemented with 10% adult sheep serum for three weeks and characterized by an antibody panel for surface markers: CD105, CD90, CD73, CD34, and CD45, before and after primary culture. Subsequently, an aliquot of cells in the first pass were cultured in a commercial human chondrogenic medium for three weeks. As a result, the percentage of surface markers for MSCs (CD105, CD90, CD73) in expanded cells in primary culture significantly increased, at the same time a decrease in the markers for hematopoietic cells (CD34 and CD45) was observed and the cells morphology was fibroblast-like. After three weeks of differentiation culture, the immunofluorescence analysis evidenced the expression of collagen-type-II. It was concluded that Mesenchymal Stem Cells isolated from mobilized peripheral blood in sheep have the ability to pre-differentiate into chondral like cells and express collagen type-II when are stimulated with a human commercial chondrogenic medium in monolayer culture.