Sunday, October 10, 2004

Osteogenic differentiation of human adipose-derived stem cells: In vitro comparison of different media and extension of survival

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: Recent studies suggest that human adipose tissue contains pluripotent stem cells similar to bone marrow-derived stem cells. These cells are easier to obtain and available in large numbers than similar cells found in bone marrow. However, the properties of ADSCs are still largely unknown. The aim of this study is to analyze the osteogenic effect and extension of life span of these differentiated PLA cells using different media’s.

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 in a control medium (Dulbecco's modified Eagle's medium + 10% fetal bovine serum) and expansion to two passages, the cells were incubated in induction Medium 1 (Dulbecco's modified Eagle's medium + 10% fetal bovine serum + dexamethasone + ascorbate-2-phosphate + Beta-glycerophosphate + antibiotics/antimycotics) and Medium 2 (Dulbecco's modified Eagle's medium + 10% fetal bovine serum + vitamin D3 + ascorbate-2-phosphate + Beta-glycerophosphate + antibiotics/antimycotics) for osteogenesis. Osteogenic differentiation was assessed at day 4 and 1, 2, 3, 4, 5, 8 and 10 weeks post-induction using von Kossa and alkaline phosphatase staining. Osteocyte specific gene expression of osteopontin, osteocalcin, and alkaline phosphatase was confirmed by RT-PCR and the release of osteocalcin was measured by enzyme-linked immunosorbent Assay (ELISA). Quantitive histomorphometry was performed with a Zeiss Axioskop II microscope and Spot software.

RESULTS: Cells placed in both osteogenic media 1 and 2 changed from flat, elongated fibroblastic nature into cuboidal forms, secreted calcified exrtracellular matrix and maintained differentiated form for more than 10 weeks. ADSCs incubated in the both osteogenic media’s stained positively for von Kossa and alkaline phosphatase. Expression of osteocyte specific genes, except osteocalcin, was also detected by RT-PCR. The amount of osteocalcin, which is a specific protein in osteoblast, increased gradually from 2 weeks till 7 weeks. Over the course of 10 weeks, there was a statistically significant increase in secretion of calcified extracellular matrix by the cells (p<0.05). No osteogenic differentiation was observed in cells incubated in the control medium.

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

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