Inhibitory Effects Of AcSDKP On Growth Of Human Bone Marrow Mesenchymal Stem Cells In Vitro

N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is a physiological regulatory peptide factor. It was originally found in fetal calf bone marrow. A body of early studies demonstrated that this acetylated tetrapeptide can inhibit the proliferation of hematopoietic stem/progenitor cells by preventing the entry of them into the S phase and arresting them in the G₀ phase. Later studies indicated that AcSDKP also has inhibitory effects on the growth of non-hematopoietic cells such as lymphocytes, renal interstitial fibroblasts and cardiac fibroblasts. Recent experiments of our laboratory showed for the first time that AcSDKP inhibited the in vitro proliferation of murine bone marrow mesenchymal stem cells (MSCs). The purpose of this study was to investigate the regulatory effects of AcSDKP on the proliferation of human bone marrow MSCs in vitro and its mechanism of action.In the present study, we have firstly isolated and purified MSCs from human bone marrow mononuclear cells, and examined their surface markers and the ability of differentiation. Subsequently, by using colony formation test, MTT colorimetric assay, mitotic index determination, 5' bromodeoxyuridine(BrdU) incorporation and flow cytometry, we have examined the proliferative activity, DNA synthesis and cell-cycle status of human bone marrow MSCs in cultures with different concentrations of AcSDKP, and evaluated the reversibility and serum concentration-dependence of its effects. We also have observed the influence of AcSDKP on the adhesive ability of these MSCs. The following results were obtained. (1) The number and size of colonies, the number of viable cells, and the mitotic index were significantly reduced in human bone marrow MSC cultures with l(T12mol/L to 10⁹mol/L AcSDKP. The maximal inhibition was observed at 10^amol/L. (2) The number of BrdU positive cells was obviously reduced in MSC cultures at 1012mol/L to 1010mol/L AcSDKP. Flow cytometric analysis indicated that cells in G0/Gi phases were markedly increased, while cells in S+G2/M phases were obviously decreased in the same cultures. (3) If AcSDKP is withdrawn following a 3-day exposure of 10 n mol/L AcSDKP to human bone marrow mononuclear cell cultures, the growth of MSCs can restore, and the MSC colonies were obviously more than those in cultures from which AcSDKP was not withdrawn. (4) The inhibition of MSC colony formation by AcSDKP had a significant decrease when the bone marrow mononuclear cells were cultured in DMEM containing 40%fetal bovine serum. (5) The number of the cells adhered to the culture plate was reduced after a 24-hour incubation of MSCs exposed to 10¹2mol/L to 10 9mol/L AcSDKP.Together, these data indicate that AcSDKP over a range of concentrations has inhibitory effects on the in vitro proliferation of human bone marrow mesenchymal stem cells. This inhibitory effect is dose-dependent and reversible. It is demonstrated that AcSDKP exerts its influence on the proliferation of MSCs by blocking or retarding the switch of quiescent MSCs into cell cycle. AcSDKP may interfere with certain stimulators of cell proliferation. Moreover, this tetrapeptide also has a possible influence on the adhesive ability of MSCs. Therefore, our work has yielded a new insight into the mechanisms of MSC growth regulation, and will provide valuable information for the use of AcSDKP in the stem cell research.