Human umbilical cord mesenchymal stem cells (UCMSCs) were isolated from umbilical cord Wharton’s jelly. Umbilical cords were obtained from the Fourth Hospital of Zhenjiang, China. Umbilical cords were rinsed twice by phosphate-buffered saline (PBS) in penicillin and streptomycin. The tissues were minced into 1–2 mm³ fragments. They were cultured in a 37°C incubator in plates with low-glucose Dulbecco’s modified Eagle’s medium (L-DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 U/ml streptomycin. The medium was changed every 3 days. After approximately two weeks, there were cells at the edge of the tissue fragments. When UCMSCs grew to 70% confluence, they were detached by 0.25% Trypsin-EDTA and reseeded in a flask of 5,000 cells/cm² for optimal proliferation. The experimental protocol was approved by the Jiangsu University ethics committee and informed consent was obtained.

UCMSCs were seeded at 1×10⁴ cells/cm² in 24-well plates with L-DMEM containing 10% FBS. After one day for attachment and growth, the medium was replaced with fresh L-DMEM supplemented with 10% FBS containing 0, 20, 40, 60, 80 and 100 μM lead acetate for 24, 48 and 72 h. Cells were harvested and counted by quantitative cytometry.

UCMSCs were grown in 96-well culture plates. After one day for attachment and growth, cells grown in 96-well plates with 200 μl L-DMDM were exposed to various concentrations of lead acetate (0, 20, 40, 60, 80 and 100 μM) for 72 h. MTT (20 μl) was added to each well for the final 4 h. When the reaction was terminated, all the solution was discarded and 150 μl DMSO was added to each well. The 96-well plate was agitated to ensure that complete solubilization of the purple formazan crystals occurred. Absorbance at 490 nm was measured using an ELISA reader.

UCMSCs were treated with lead acetate for 24, 48 and 72 h. Cells were harvested and washed twice with PBS and stained with propidium iodide (PI) (Sigma-Aldrich, USA) for 30 min in dark conditions. The stained cells were analyzed by flow cytometry.

UCMSCs were treated with lead acetate for 48 h. Following treatment, the cells were trypsinized with 0.25% trypsin-EDTA, washed twice with PBS and stained with PI and annexin-V-FITC according to the manufacturer’s instructions. The stained cells were analyzed by flow cytometry.

UCMSCs were seeded at a density of 3×10⁴ cells/cm² and stimulated with osteogenic induction medium for 14 days. The osteogenic medium consisted of low-glucose DMEM, 10% FBS, 100 nM dexamethasone, 10 mM sodium β-glycerophosphate and 0.05 mM-ascorbic acid 2-phosphate (all from Sigma), and was replaced every 3 days.

In the differentiated UCMSCs, osteogenic characteristics were confirmed by alkaline phosphatase (ALP) expression using histochemical staining and the observation of the hydroxyapatite matrix using Von Kossa staining. Cells were fixed with fixing agent and stained with ALP according to the manufacturer’s instructions. The Von Kossa stain was performed according to the protocol described previously (23,24) with a few modifications. The cells were fixed with 4% paraformaldehyde for 5 min at room temperature, stained for 20–60 min with 2% silver nitrate (Sigma-Aldrich) and then treated with 5% sodium thiosulfate (Sigma-Aldrich) for 2 min.

Total RNA was extracted from UCMSCs and UCMSCs treated with lead acetate using TRIzol reagent (Invitrogen, USA). cDNA was obtained from 1 μg total RNA using an oligo-dT primer in a reaction volume of 20 μl, using a reverse transcription kit according to the manufacturer’s instructions (Fermentas). The cDNA samples were subjected to polymerase chain reaction (PCR) using specific primers. The conditions of PCR were as follows: Initial denaturation at 94°C for 5 min, denaturation at 94°C for 30 sec, annealing at 55–70°C for 30 sec (see Table I for temperatures used), extension for 30 sec at 72°C for 30 cycles, and a final polymerization at 72°C for 10 min. PCR products were analyzed by electrophoresis on a 1.5% agarose gel with ethidium bromide staining and photographed under UV transillumination. β-actin was used as a control. The specific primers were designed as shown in Table I.

UCMSCs were seeded in a 6-well plate with L-DMEM medium supplemented with 10% FBS. After one day for attachment and growth, the medium was replaced with fresh L-DMEM supplemented with 10% FBS containing 60 μM lead acetate for 72 h. There was no lead acetate in the control group. Following treatment, the medium was removed and cells were washed with PBS 2–3 times. Sterile 3% agar was prepared and incubated at 65°C in a waterbath. Bone marrow cells were added to a 10 ml tube with 9 ml L-DMEM medium supplemented with 10% FBS and incubated at 37°C. In total, 1 ml 3% agar was added to 9 ml cell suspension and mixed gently by swirling, and 1 ml was quickly added to each of the wells of the prepared 6-well plate. Plates were incubated at 37°C for 14 days. Colonies were counted using a microscope.

UCMSCs were exposed to various concentrations of lead acetate (0, 20, 40, 60, 80 and 100 μM) for 24, 48 and 72 h, and counted with a cytometer. The number of cells was significantly decreased in a dose-dependent manner following lead acetate treatment for 24, 48 and 72 h (Fig. 1A). To further investigate the cytotoxic effects of lead acetate on cell proliferation, we used the MTT assay to test lead acetate-induced toxicity in UCMSCs. After 72 h of lead acetate treatment, cell viability was markedly decreased in a dose-dependent manner (Fig. 1B).

Treatment of lead acetate at the concentration of 60 μM showed an increase in annexin V-positive cells over the untreated UCMSCs (Fig. 2), which suggested that lead acetate is capable of inducing apoptosis in UCMSCs.

In order to investigate the effect of lead acetate on the differentiation potential, UCMSCs were induced to osteogenic differentiation. Following osteogenic differentiation of UCMSCs, the cells were stained with ALP and Von Kossa. Compared with the control, the rates of ALP-positive staining and extracellular matrix (ECM) calcium deposits were reduced following lead acetate treatment (Fig. 3). The gene expression of ALP also revealed a marked downregulation in the experimental group (Fig. 3E). These results indicated that lead acetate inhibited the osteogenic differentiation of MSCs.

To investigate the effect of lead acetate on the hematopoietic system, the mRNA expression of cytokines was detected in UCMSCs. These cytokines are an important component of the hematopoietic microenvironment as well as MSCs. UCMSCs were treated with lead acetate (60 μM) and RT-PCR was performed to analyze mRNA expression. Following lead treatment, certain genes which were connected with the hematopoietic system and angiopoiesis, including Ang-1, Ang-2, VEGF, SCF, Flt3-ligand and TPO, were significantly decreased (Fig. 4). These results indicated that lead acetate could induce hematopoietic system damage through inhibiting the hematopoiesis-supportive function of MSCs.

MSCs have a hematopoiesis-supportive function in vitro. To further prove the reverse effect of lead acetate on the hematopoiesis-supportive function of MSCs, we detected the supportive function of UCMSCs on the colony formation ability of bone marrow cells following treatment of lead acetate. After 7, 10 and 14 days, we counted the number of colonies in each well and found that the number of cells in the experimental groups were all significantly lower than that of the control group (Fig. 5 and Table II). Following treatment with lead, the supportive function of MSCs on colony formation was inhibited.