Isolation of adipose-derived stem cells and their induction to a chondrogenic phenotype

Abstract

The ability to isolate, expand and differentiate adult stem cells into a chondrogenic lineage is an important step in the development of tissue engineering approaches for cartilage repair or regeneration for the treatment of joint injury or osteoarthritis, as well as for their application in plastic or reconstructive surgery. Adipose-derived stem cells (ASCs) provide an abundant and easily accessible source of adult stem cells for use in such regenerative approaches. This protocol first describes the isolation of ASCs from liposuction aspirate. The cell culture conditions provided for ASC expansion provide a large number of multipotent stem cells. Instructions for growth factor-based induction of ASCs into chondrocyte-like cells using either cell pellet or alginate bead systems are detailed. These methods are similar to those published for chondrogenesis of bone marrow-derived mesenchymal stem cells but distinct because of the unique nature of ASCs. Investigators can expect consistent differentiation of ASCs, allowing for slight variation as a result of donor and serum lot effects. Approximately 10-12 weeks are needed for the entire process of ASC isolation, including the characterization of chondrocyte-like cells, which is also described.

Figure 1

Flow chart for experiments with associated timing

Figure 2

Expansion of ASCs in expansion medium over 5 days. Appearance of cells at 10x after (a) 30 minutes, (b) 3 hours, (c) 1 day, (d) 2 days, (e) 3 days, (f) 4 days, and (g) 5 days. Black arrows in (h) and (i) show cells with atypical or abnormal morphology. Scale bar = 100 mm.

Figure 3

2.5x microscope image (2.5x) of ASCs encapsulated in alginate. Scale bar = 1 mm.

Figure 4

Toluidine Blue stain on (a) human articular cartilage and (b) and (c) are representative images of typical variation in GAG synthesis and accumulation obtained from ASC pellets cultured for 14 days. (Note. Expect similar results if Safranin-O stain were used.) Scale bars = 100 mm.

Figure 5

Representative immunohistochemistry results for chondroitin-4-sulfate and types I, II, and X collagen for a typical experiment with ASCs encapsulated in alginate after 4 weeks in in vitro culture. Positive Control: porcine cartilage for C-4-S, Collagen II, and Collagen X. Porcine ligament for Collagen 1. Control: incomplete chondrogenic medium supplemented with 10% FBS. Treatment: incomplete chondrogenic medium supplemented with short term exposure to BMP-6 in addition to continuous exposure to rhEGF, rhFGF, and 10% FBS. Scale bars = 20 mm. Figure modified from Diekman, B.O., Estes, B.T. & Guilak, F. The effects of BMP6 overexpression on adipose stem cell chondrogenesis: Interactions with dexamethasone and exogenous growth factors. J Biomed Mater Res A (2009). Reprinted with permission from John Wiley and Sons.

Figure 6

Pellet size after 6 weeks of culture. (a) representative image of pellets in 15 ml conical tubes in incomplete chondrogenic medium + 10% FBS on the left and complete chondrogenic medium containing TGF-b on the right and (b) the left two pellets were cultured in incomplete chondrogenic medium + 10% FBS and the right two pellets were cultured in complete chondrogenic medium containing TGF-b. Scale bar = 1 mm.

Figure 7

Typical DMB (A) and OH-Proline (B) assay standard curves. n=3 wells per concentration. Data are mean values ± SEM.