Improved Isolation of Mesenchymal Stem Cells Based on Interactions between N-Acetylglucosamine-Bearing Polymers and Cell-Surface Vimentin

Abstract

Mesenchymal stem cells (MSCs) in bone marrow and adipose tissues are expected to be effective tools for regenerative medicine to treat various diseases. To obtain MSCs that possess both high differentiation and tissue regenerative potential, it is necessary to establish an isolation system that does not require long-term culture. It has previously been reported that the cytoskeletal protein vimentin, expressed on the surfaces of multiple cell types, possesses N-acetylglucosamine- (GlcNAc-) binding activity. Therefore, we tried to exploit this interaction to efficiently isolate MSCs from rat bone marrow cells using GlcNAc-bearing polymer-coated dishes. Cells isolated by this method were identified as MSCs because they were CD34-, CD45-, and CD11b/c-negative and CD90-, CD29-, CD44-, CD54-, CD73-, and CD105-positive. Osteoblast, adipocyte, and chondrocyte differentiation was observed in these cells. In total, yields of rat MSCs were threefold to fourfold higher using GlcNAc-bearing polymer-coated dishes than yields using conventional tissue-culture dishes. Interestingly, MSCs isolated with GlcNAc-bearing polymer-coated dishes strongly expressed CD106, whereas those isolated with conventional tissue-culture dishes had low CD106 expression. Moreover, senescence-associated β-galactosidase activity in MSCs from GlcNAc-bearing polymer-coated dishes was lower than that in MSCs from tissue-culture dishes. These results establish an improved isolation method for high-quality MSCs.

Figure 1

Evaluation of AC-GlcNAc coatings. (a) Structure of AC-GlcNAc. (b) Evaluation of AC-GlcNAc coating on polystyrene dishes using the interaction between biotinylated sWGA and AC-GlcNAc. The amount of biotinylated sWGA on dishes was measured by HRP-neutravidin and TMB solution (absorbance at 450 nm). (c) Western blots demonstrating expression of cell-surface vimentin in human immortalized MSCs. (d) UE7T-13 adherence to AC-GlcNAc-coated dishes at 37 (left) and 4°C (right). ^∗P < 0.01, n = 3.

Figure 2

The interaction between cell-surface vimentin and AC-GlcNAc is responsible for the adhesion of UE7T-13 cells to coated dishes. (a) Inhibition of adhesion by AC-GlcNAc at 37 (left) and 4°C (right), n = 4. (b) Expression of vimentin in knockdown and control cells. (c) Inhibition of adhesion by vimentin knockdown. Adherence of vimentin-knockdown and control cells to AC-GlcNAc 5-coated dishes at 4°C, n = 5. ^∗P < 0.01.

Figure 3

Characteristics of rat bone marrow cells grown on AC-GlcNAc-coated and uncoated tissue-culture dishes. (a) Flow cytometric analysis of expression of cell-surface vimentin in rat bone marrow cells. (b) Phase-contrast images of colonies formed on tissue-culture dishes and AC-GlcNAc-coated dishes at 10 days. Lower images are enlargements of upper images. (c) Images of crystal violet-stained colonies and areas of colonies over 3 to 16 days of culture. ^∗P < 0.01, n = 3.

Figure 4

Colony formation of rat bone marrow cells on AC-GlcNAc-coated dishes and tissue-culture dishes. (a) Representative images and areas of colonies after 17 days of culture. ^∗P < 0.01, n = 18. (b) Representative images and areas of colonies on AC-GlcNAc-coated dishes, PV-MA-coated dishes, and tissue-culture dishes for 10 days. ^∗P < 0.01, n = 3.

Figure 5

Flow cytometric analysis of proliferating cells from AC-GlcNAc-coated and control uncoated dishes. (a) Representative charts of flow cytometric analysis for expression of MSC markers (CD34, CD45, CD11b/c, CD90, CD29, CD44, CD54, CD73, CD105, and CD106) in the proliferating cells on each dish. (b) Percentages of cells positive for MSC-positive and MSC-negative markers.

Figure 6

AC-GlcNAc-selected cells have reduced levels of the senescence marker SA-β-galactosidase. (a) Representative chart of flow cytometric analysis of SA-β-galactosidase activity. (b) SA-β-galactosidase activity.

Figure 7

Osteogenic, adipogenic, and chondrogenic differentiation is increased by selection of cells adhering to AC-GlcNAc-coated dishes.