Effects of insulin-like growth factor-1 on the properties of mesenchymal stem cells in vitro

Abstract

Objective: To explore the effects of insulin-like growth factor-1 (IGF-1) on migration, proliferation and differentiation of mesenchymal stem cells (MSCs).

Methods: MSCs were obtained from Sprague-Dawley rats by a combination of gradient centrifugation and cell culture techniques and treated with IGF-1 at concentrations of 5-20 ng/ml. Proliferation of MSCs was determined as the mean doubling time. Expression of CXC chemokine receptor 4 (CXCR4) and migration property were determined by flow cytometry and transwell migration essay, respectively. mRNA expression of GATA-4 and collagen II was determined by reverse transcription-polymerase chain reaction (RT-PCR).

Results: The mean doubling time of MSC proliferation was decreased, and the expression of CXCR4 on MSCs and migration of MSCs were increased by IGF-1, all in a dose-dependent manner, while the optimal concentration of IGF-1 on proliferation and migration was different. IGF-1 did not affect the expression of GATA-4 or collagen II mRNA.

Conclusions: IGF-1 dose-dependently stimulated the proliferation of MSCs, upregulated the expression of CXCR4, and accelerated migration. There was no apparent differentiation of MSCs to cardiomyocytes or chondrocytes after culturing with IGF-1 alone.

Fig. 1

Expression of cell markers in MSCs MSCs at the fourth passage were trypsinized and diluted by PBS to a concentration of 1×10⁶ cells/ml and then incubated with mouse anti-rat CD34, CD45, CD29 or CD44 antibodies (1:500), followed by FITC-labeled goat-anti-mouse IgG (1:200) for 45 min at 4 °C. Fluorescent intensity was detected by flow cytometry. Each group was assessed in triplicate. (a) CD34, (0.28±0.12)%; (b) CD45, (1.98±0.43)%; (c) CD29, (96.34±2.15)%; (d) CD44, (78.64±3.26)%

Fig. 2

Growth curves for MSCs cultured with or without IGF-1 MSCs at the fourth passage were trypsinized, replaced and cultured with 5, 10 or 20 ng/ml IGF-1 for 7 d. The number of cells in each well was counted using a hemocytometer and cell growth curves were drawn. The growth curve for each group showed similar ‘S’ shape. The first 2 d represented the latent phase. The logarithmic growth phase was from Days 3 to 6. Days 7–8 represented the plateau phase

Fig. 3

Expression of CXCR4 in MSCs After culture, the MSCs were trypsinized and diluted in PBS. Mouse anti-rat CXCR4 (1:200) and FITC-labeled goat-anti-mouse IgG (1:200) were added to the cell suspension and incubated for 45 min at 4 °C. Fluorescent intensity was detected by flow cytometry. (a) Control group, (10.38±1.29)%; (b) IGF-1 5 ng/ml group, (18.35±2.68)%; (c) IGF-1 10 ng/ml group, (28.62±3.42)%; (d) IGF-1 20 ng/ml group, (38.94±3.93)%

Fig. 4

MSC migration assay by transwell The number of cells migrated through pores was counted under 10 random high power fields for each group. IGF-1 enhanced the migratory ability of MSCs in a dose-dependent manner. (a–d) Pictures of migrated MSCs: (a) Control group; (b) IGF-1 5 ng/ml group; (c) IGF-1 10 ng/ml group; (d) IGF-1 20 ng/ml group. (e) Average number of migrated cells in each group. ^† P<0.05 vs. control group; ^* P<0.05 vs. IGF-1 5 ng/ml group; ^# P<0.05 vs. IGF-1 10 ng/ml group

Fig. 5

Expression of GATA-4 and collagen II mRNA in MSCs cultured with IGF-1 The expression of markers for chondrocytes (collagen II) and cardiomyocytes (GATA-4) was assessed by RT-PCR. Cardiomyocytes and chondrocytes were used as positive controls for GATA-4 and collagen II, respectively. GAPDH was used as internal control. The PCR products for collagen II, GATA-4 and GAPDH were 518, 179 and 280 bp, respectively. Lane 1: DNA ladder DL2000 (bp); Lane 2: control group; Lane 3: IGF-1 5 ng/ml group; Lane 4: IGF-1 10 ng/ml group; Lane 5: IGF-1 20 ng/ml group; Lane 6: chondrocytes; Lane 7: cardiomyocytes; Lane 8: negative control