Differentiation of mesenchymal stem cells into myocardial cells promoted by stem cell factors

Cuiyu BAO; Jun GUO; Yexin MA; Min ZHENG

ABSTRACT

BACKGROUND:

Mesenchymal stem cells (MSCs) can differentiate into myocardial cells in vitro and in vivo, but the amount is small and directed differentiation rate is low.

OBJECTIVE:

To explore the effect of stem cell factor (SCF) on promotion of MSCs differentiating into myocardial cells.

DESIGN:

Opening experiment.

SETTING:

Institute of Cardiovascular Disease, Xianning College and Department of Cardiology, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology.MATERIALS The experiment was performed at the Experimental Center of Institute of Cardiovascular Disease, Xianning College from October 2003to August 2004. A total of 20 infant SD rats aged 1-2 days were selected for culture of myocardial cells. Another 25 clean adult SD rats were selected and randomly divided into SCF group, blank control group with 12 rats in each group. The left one rat was used for MSCs extraction, culture and purification.

METHODS:

①MSCs were labeled with 4', 6-diamidino-2-phenylindole,dihydrochloride (DAPI) before co-culture. SCF was given into rats of the SCF group by subcutaneous injection successively for 5 days, 20 μg/kg per day. Isolated MSCs were co-cultured with myocardial cells that had been cultured for 3 days. Saline of the same volume was given in the blank control group by subcutaneous injection. MSCs without any intervention were co-cultured with myocardial cells that had been cultured for 3 days.②Expressions of MHC α/β and troponin T were recorded and measured with digital micro-camera shot and immunofluorescence technique, respectively. Percentage of DAPI labeled MSCs differentiating into myocardial cells was measured.MAIN OUTCOME

MEASURES:

①Growth of MSCs, ②detection of labeling rate of DAPI on MSCs, ③analysis of activity and purity of co-cultured myocardial cells, and ④differentiation of MSCs into myocardial cells after co-culture.

RESULTS:

①Bone marrow cell suspension was inoculated in plastic petri dish. Round cells scattered at the bottom of bottle. At hour 24 a few long fusiform shape adhered cells appeared following liquor change, and at day 4 many fusiform shape adhered cells appeared, which entered logarithm increased period. At days 8-12 80% were confluence. The proliferation of passage cells was more rapid. With liquor change and passage, the MSCs were purified gradually. ②Labeling rate of DAPI on MSCs was 100%. ③At 3-day in vitro culture of myocardial cells the percentage of beat cells was 63%, with beat frequency of 40-60 times per minute. Percentage of positive cells of troponin T expression was 75% examined with immunocytochemical technique. ④At co-cultured days 2 and 3, positive percentage of DAPI labeled MSCs expressing MHC α/β was significantly higher in the SCF group than in the blank control group (P ＜ 0.01). At the co-cultured days 3, 4 and 5, positive percentage of DAPI labeled MSCs expressing troponin T was obviously higher in the SCF group than in the blank control group (P ＜ 0.01).

CONCLUSION:

SCF has markedly accelerating effect on proliferation and differentiation of MSCs.