Differentiation from rat bone marrow stromal stem cells into cardiomyocytes induced by 5-azacitidine in vitro

Fengxiang ZHANG; Shuqing LI; Dapeng SUN; Zhongyi SI

ABSTRACT

BACKGROUND:

Bone marrow stromal stem cells (BMSCs) are characterized by rapid amplification and wide differentiation.Thus, to establish in vitro BMSC induction models contributes to the study of tissue engineering.

OBJECTIVE:

To investigate the possibility of inducing the differentiation of rat BMSCs into cardiomyocytes by 5-azacitidine in vitro.OESING, TIME AND

SETTING:

The cytological in vitro study was performed at the Central Laboratory of First Affiliated Hospital of Liaoning Medical University from June 2005 to June 2008.MATERIALS A total of 20 Sprague Dawley rats were supplied by the Experimental Animal Center, Liaoning Medical University.5-azacitidine (Sigma, USA) was used.MHEHODS Following anesthesia, the rats were used to isolate the femur and tibia. BMSCs were isolated and cultured by the whole bone marrow method + adherent method. When 90% BMSCs were confluence, BMSCs were passaged. BMSCs at the third passage were incubated in a 24-well plate at 2×10~4/well, with 5,10, 15, 20 μmol/L 5-azacitidine. Simultaneously, a blank control group (without inductor) was set. Following 24 hours of induction, BMSCs were incubated in normal medium for 3 weeks.MAIN OUTCOME

MEASURES:

The following parameters were measured cell appearance and growth curve, morphological changes following induction, and expression of connexin-43 and a-striated muscle actin.

RESULTS:

Cultured BMSCs were spindle-shape, with some cell confluence. P3 cells following incubation entered static phase at 1 and 2 days, and entered logarithmic phase at 3 days, reached a peak at 9 days, and then entered platform stage. Cell number became decreased at 12 days. Following induction of 5 μmol/L 5-azacitidine, no significant difference was found in BMSCs.Following induction of 10 μmol/L 5-azacitidine, BMSCs became long and big, extended towards a direction, with the property of myotube formation cells. Following induction of 15 umol/L 5-azacitidine, a few cells survived surrounding the 24-well plate.Following induction of 20 μmol/L 5-azacitidine, cells died. Following induction of 10 μmol/L 5-azacitidine for 3 weeks, expression of connexin-43 and a-striated muscle actin was determined in BMSCs. However, a negative expression was detected in the blank control group.

CONCLUSION:

BMSCs cannot differentiate into cardiomyocytes by itself. Following in vitro induction, BMSCs can differentiate into cardiomyocytes. Low-dose 5-azacitidine concentration cannot induce the differentiation, but high-dose 5-azacitidine concentration will induce death in a large number of cells. Thus, 10 μmol/L is an optimal concentration.