Differentiation of umbilical cord blood mesenchymal stem cells into osteoblasts in vitro

Jingjie TONG; Shan XU; Heng LV; Qiang CHU

ABSTRACT

BACKGROUND:

Umbilical cord blood mesenchymal stem cells (UCB-MSCs) can differentiate into various types of cells under certain condisions, and easily proliferate in vitro. However, UCB-MSCs have long establishment time and low frequency.

OBJECTIVE:

To in vitro isolate and culture UCB-MSCs, and induce its differentiation into osteoblasts.DESIGN, TIM E AND

SETTING:

The in vitro cytological study was performed at the Laboratory of the Medical College of Qingdao University from June 2008 to January 2009.MATERIALS UCB was obtained from term normal delivery women at the Department of Gynaecology and Obstetrics, Qingdao Municipal Hospital.

METHODS:

Human UCB-MSCs were isolated and cultured in vitro by Percoll density gradient. When reached 90% confluency,UCB-MSCs were digested by trypsin for subculture. At the third passage, UCB-MSCs at 1×106 were incubated. When reached 50% 60% cenfluency, UCB-MSCs were treated with DMEM supplemented with 0.1 μmol/L dexamathasone, 10 mmol/Lβ-sodium glycerophosphate and 50 μmol/L vitamin C. UCB-MSCs in the control group were incubated in low glucose DMEM.MAIN OUTCOME

MEASURES:

Growth and proliferation of MSCs were observed under the inverted microscope. Cell surface marker expression and cell growth curve were measured by flow cytometry. Cell ultrastructure was observed under the transmission electron microscope. Differentiation of UCB-MSCs into osteoblasts was determined by Won Kossa staining and alkaline phosphatase staining.

RESULTS:

Primary cultured UCB-MSCs had similar morphology to bone marrow mesenchymal stem cells. After passage, cell morphology was even, presenting spindle shape. UCB-MSCs at passage 3 highly expressed CD29, CD44, CD13, but did not express CD34. Growth latency was 2-3 days. Cells entered logarithm proliferation phase at days 3-4, and platform phase 1 month later. Nuclei presented round or irregular, with clear nuclear membrane, 1-2 nucleoli, rough chromatin, abundant organelles and microvilli. UCB-MSCs at passage 3 were gradually confluent following 3 days of osteogenic induction, with the presence of pavement-stone shape. 14 days later, calcified nodules by Von Kossa staining, and cells were positive for alkaline phosphatase staining. In the control group, no calcified nodules were found, and cells were negative for alkaline phosphatase staining.

CONCLUSION:

UCB-MSCs can be successfully isolated by Percoil density gradient, and induced to differentiate into osteoblasts in vitro.