J Korean Foot Ankle Soc.  2014 Sep;18(3):100-107. 10.14193/jkfas.2014.18.3.100.

Comparison of Efficiency of Self-renewal and Differentiation Potential in Tendon-derived Mesenchymal Stem Cells Isolated by Magnetic-activated Cell Sorting Method or Colony Picking Method

• Lee M
• Choi Y
• Yoon DS
• Lee JW
• Yoon GS
• Choi WJ
• Han SH

• KMID: 2333472
• DOI: http://doi.org/10.14193/jkfas.2014.18.3.100

Abstract

PURPOSE
The purpose of this study is to evaluate the efficacy of mesenchymal stem cell (MSC) isolation by the magnetic-activated cell sorting (MACS) method in tendon tissue-derived cells compared to the colony picking method for isolation of MSCs by picking colonyforming cells.
MATERIALS AND METHODS
Human tendon-derived cells were isolated by enzyme digestion using normal tendon tissues from three donors. We used the magnetic kit and well-known MSC markers (CD90 or CD105) to isolate MSCs in tendon-derived cells using MACS. Cloning cylinders were used to isolate colony-forming cells having MSC characteristics in tendon-derived cells. Colony-forming unitfibroblast (CFU-F) assay was used to evaluate the self-renewal capacity of cells isolated using the colony picking method or MACS. For comparison of differentiation potentials into osteogenic or adipogenic lineage between two groups, alizarin red S and oil red O staining were performed at 14 days after induction of differentiation in vitro.
RESULTS
Flow cytometry results showed that early passage tendon-derived cells expressed CD44 in 99.13%, CD90 in 56.51%, and CD105 in 86.19%. In the CFU-F assay, CD90+ or CD105+ cells isolated with MACS showed larger colony formation in size than cells isolated using the colony picking method. We also observed that CD90+ or CD105+ cells were constantly differentiated into both osteogenic and adipogenic lineages in cells from all donors, whereas cells isolated using the colony picking method were heterogeneous in differentiation potentials to the osteogenic and adipogenic lineages.
CONCLUSION
CD90+ or CD105+ cells isolated using MACS showed superior MSC characteristics in the self-renewal and multi-differentiation capacities compared with cells isolated using the colony picking method.

Keyword

Tendons; Mesenchymal stem cells; Magnetic-activated cell sorting; Colony picking method

MeSH Terms

Clone Cells
Cloning, Organism
Digestion
Flow Cytometry
Humans
Mesenchymal Stromal Cells*
Tendons
Tissue Donors

Figure

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  Figure 1. Identification of mesenchymal stem cell surface marker CD44, CD90, and CD105 in tendon-derived cells. Flow cytometry analysis of the expression of cell surface markers related to mesenchymal stem cells such as CD44, CD90, and CD105 was performed in tendon-derived cells at passage 2.

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  Figure 2. Isolation methods for tendon-derived mesenchymal stem cells. (A) The cell colonies were dissociated by cloning cylinder and each colony was cultured in growth medium (upper). The isolation method using mesenchymal stem cell surface markers CD90 and CD105 was performed by magnetic kit (bottom). (B) The cells isolated by magnetic-activated cell sorting using CD90 and CD105 were analyzed by flow cytometry.

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  Figure 3. Colony forming capacity. Total colonies were stained with crystal violet at 12 days.

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  Figure 4. Quantification of colony-forming units. The number of large colonies was counted and the percentage of large colonies was measured.

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  Figure 5. Osteogenic differentiation capacity. Each cell was seeded on 12-well plates and differentiated for 14 days. (A) The cells isolated by each method were determined by alkaline phosphatase staining at 7 days. Scale bars=200 μm. (B) After induction of osteogenic differentiation for 14 days, alizarin red S staining was performed. (C) Absorbance at 595 nm was measured with a spectrophotometer for quantitative analysis of the stained mineralized nodules.

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  Figure 6. Adipogenic differentiation capacity. (A) After induction of adipogenic differentiation for 14 days, lipid droplets were detected by oil red O staining. Scale bars=200 μm. (B) For quantification of the stained lipid droplets, absorbance at 500 nm was measured with a spectrophotometer.

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