Download Osteogenic differentiation by human adipose

Title:
Osteogenic differentiation of human adipose-derived stem cells:
In vitro comparison of
different media and extension of survival.
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: 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 & 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 +
-
glycerophosphate + antibiotics/antimycotics); Medium 2 (Dulbecco's modified Eagle's medium + 10%
fetal
bovine
serum + vitamin
D3 + ascorbate-2-phosphate +
-glycerophosphate
+
antibiotics/antimycotics); and Medium 3 (Dulbecco's modified Eagle's medium + 10% fetal bovine
serum + vitamin
D3 + dexamethasone
+
ascorbate-2-phosphate +
-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
RT-PCR and
was confirmed by
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 all osteogenic media 1, 2 and 3 changed from flat, elongated fibroblastic
nature into cuboidal forms, secreted calcified exrtracellular matrix and maintained differentiated form
for more than 10 weeks (Fig. 1). ADSCs incubated in all the 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.
Von Kossa Staining
Alkaline Phosphatase Staining
Figure 1. Positive staining for alkaline phosphatase and von kossa post 10 weeks culture of PLA cells
in bone differentiation media.