Sunday, October 10, 2004

Long term in vitro chondrogenic differentiation of human adipose-derived stem cells

Eul-Sik Yoon, MD, PhD, Hooman Shabatian, MD, Sanjay Dhar, PhD, Michael P. McConnell, MD, Jay W. Calvert, MD, and Gregory R.D. Evans, MD, FACS.

INTRODUCTION: Multipotential processed lipoaspirated (PLA) cells from human liposuctions have been induced into the chondrogenic phenotype using chondrogenic media. Isolation of single cell populations of PLA cells has shown that they have at least potential to form bone, cartilage, and fat. Compared with cells harvested by bone-marrow aspiration, PLA cells are easier to obtain, have lower donor-site morbidity, and are available in larger numbers which eliminates the need for costly and lengthy tissue culture expansion. Our aim was to determine if the PLA cells have the potential for induction into the chondrogenic phenotypes over an extended period of time than reported in literature.

MATERIALS AND METHODS: Human Adipose-derived stromal cells (ADSCs) were isolated from lipoaspirates after extensive washing with phosphate-buffered saline and treatment with collagenase. After primary culture and expansion to two passages in control medium (Dulbecco's modified Eagle's medium + 10% fetal bovine serum), the cells were cultured using modified micromass technique in chondrogenic medium which consisted of DMEM-1 supplemented with 6.25 ug/ml insulin, 10ng/ml TGFb1, 50nM Ascorbic acid-2-phosphate and 1% antibiotic/antimycotic for 12 weeks. Chondrogenesis was analyzed by Alcian blue staining and collagen type II immunohistochemistry. Chondrogenic differentiation was evaluated by histological, biochemical, and RNA analyses for the expression of cartilage extracellular matrix components. The multiple experimental time points for chondrogenic differentiation were studied at week 1, 2, 3, 4, 8 and 12 after standardizing each experimental group for cell number.

RESULTS: PLA cells with chondrogenic media condensed into white small spheroids or round structures measuring approximately 1 to 2 mm in diameter that were visible to the naked eye within 24 hours of induction. The nodule stained positively for alcian blue at pH 1.0 and type II collagen. Over the course of 10 weeks, the number of cartilage nodules increased but there no increase in nodule size. Plating at increasing cell densities resulted in the formation of more cartilaginous nodules. No nodules were observed in non-chondrogenic control or chondrogenic media in monolayer cultures. RT-PCR analysis confirmed the expression of type II collagen and the cartilage-specific proteoglycan-aggrecan.

CONCLUSION: Adipose tissue-derived stromal cells, sometimes called as processed lipoaspirate (PLA) cells from human have chondrogenic potential in vitro for longer duration than previously published. ADSC may be an ideal source for further experiments on stem cell biology and regenerative medicine.

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