Chondrogenic differentiation of bone marrow-derived mesenchymal stem cells: tips and tricks

Abstract

It is well known that adult cartilage lacks the ability to repair itself; this makes articular cartilage a very attractive target for tissue engineering. The majority of articular cartilage repair models attempt to deliver or recruit reparative cells to the site of injury. A number of efforts are directed to the characterization of progenitor cells and the understanding of the mechanisms involved in their chondrogenic differentiation. Our laboratory has focused on cartilage repair using mesenchymal stem cells and studied their differentiation into cartilage. Mesenchymal stem cells are attractive candidates for cartilage repair due to their osteogenic and chondrogenic potential, ease of harvest, and ease of expansion in culture. However, the need for chondrogenic differentiation is superposed on other technical issues associated with cartilage repair; this adds a level of complexity over using mature chondrocytes. This chapter will focus on the methods involved in the isolation and expansion of human mesenchymal stem cells, their differentiation along the chondrogenic lineage, and the qualitative and quantitative assessment of chondrogenic differentiation.

Fig. 1

Bone marrow aspirate in the syringe immediately after aspiration from a healthy volunteer donor.

Fig. 2

Loose bone marrow cell pellet after initial centrifugation.

Fig. 3

Bone marrow cells being layered over the preformed Percoll gradient.

Fig. 4

Sample after gradient fractionation; note the band of nucleated cells at the top of the gradient.

Fig. 5

Pellet of mononuclear cells after the final wash and centrifugation.

Fig. 6

Appearance of hMSC colonies in primary culture. (a) Control culture. (b) FGF-treated culture; note that the colonies are looser and there is a higher number of cells between the colonies in the FGF-treated cultures. Crystal black staining.

Fig. 7

Appearance of hMSC colonies in primary culture. (a) Colonies are not yet ready to be subcultured. (b) Colonies must be subcultured; note the higher cell density in the center of the colonies. Crystal black staining.

Fig. 8

Comparison of plasticware and incubator space needed for 200 aggregates in 15-ml centrifuge tubes or in 96-well polypropylene plates.

Fig. 9

MSC aggregate after 3 weeks in chondrogenic conditions. Toluidine blue staining.

Fig. 10

Immunohistochemical staining of serial sections of a 3-week aggregate. Type I collagen (left), type II collagen (middle), and type X collagen (right). Note the absence of type I collagen staining throughout the aggregate and the lack of type X staining in the periphery of the aggregate. The contours of the aggregates have been outlined to help identify areas of the aggregates devoid of specific types of collagen.

Fig. 11

Assembled dot-blot apparatus.

Fig. 12

Dots being punched from the membrane and transferred to the microcentrifuge tubes.

Fig. 13

Homogenizer tip and 4-ml cryovial used for the RNA isolation.