Osteogenic Differentiation of Human PLA Cells in Vitro and Vivo

Title: Long term in vitrochondrogenic differentiation of human adipose-derived stem cells

Authors: Eul-Sik Yoon, MD, PhD, Hooman S. 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 chondrogenicphenotype using chondrogenic media.Isolation of single cell populations of PLA cells has shown that they have atleast 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 determineif 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 ㎍/ml insulin, 10ng/ml TGFβ1, 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 differentiationwere studied atweek 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 spheroidsor 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 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.

Chondrogenic differentiation of PLA derived stem cells

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Figure1: Induced chondrogenic potential of PLA derived stem cells as shown by cartilage-specific collagen type II staining (A) and Alcian blue staining (B) in micromass conditions at week 4 post induction.