The Biomarkers And Variation With Adipogenic Differentiation Of Mesenchymal Stem Cells Studied By In Vitro 9.4T MR Spectroscopy

Objective:To study the biomarkers of the human mesenchymal stem cells (MSCs) isolated from umbilical cord Wharton's Jelly and observe the variation of the biomarkers as quantitative analysis with adipogenic differentiation of MSCs by using in vitro 9.4T high resolution magnetic resonance (MR) spectroscopy.Methods:When the MSCs and the EC109 cell line grew up to 80-85% confluence at passage 4, they were collected (about 7-8×106, samples were n=6 and n=2; the primary cells were presented by Multidisciplinary Study Center and Pathology Laboratory of Shantou respectively). The MSCs were underwent adipogenic differentiation for 14days in the 10% FBS/LG-DMEM culture medium with 1μM dexamethasone,500μM 3-isobutyl-1-methylxanthine(IBMX),60μM indomethacin and 5μM insulin (samples was n=7). At the 14th day, the cells undergoing adipogenic differentiation were assessed by using the Red-0 staining and Reverse Transcription-Polymerase Chain Reaction (RT-PCR).1H-MRS data acquisitions were performed by using in vitro 9.4T high resolution MR spectroscopy (Bruker Avance 400MHz). Spectra was primarily processed in the Frequecvy domain with the XWINNMR (Bruker GmBH) and then analyzed with Mestre-c 4.7.Results:The spectrums of the MSCs were high quality and good reproducibility. Several major metabolites can be observed in the MSCs spectra, including choline compound, creatine, glutamate, myo-inositol, acetate, succinate, methionine and some fatty acids (1.28 ppm biomarker, which was considered as the unique for the neural progenitor cells). Quantification of metabolite concentrations was performed, and the levels of intracellular metabolites, such as choline compounds, creatine, glutamate and acetate all decreased, with the increased level of methionine, succinate and fatty acids after the MSCs differentiation 2 weeks. Intracellular choline, acetate, glutamate and creatine reduced from 6.3±0.68, 0.97±0.23,0.3±0.05 and 0.1±0.02 mM to 1.1±0.06 (p<0.01),0.45±0.1 (p<0.01),0.16±0.08 mM (p<0.05) and no-detected, respectively. Inversely, the methionine, succinate and fatty acids (1.28 ppm) increased from 0.03±0.01,0.11±0.02, (1.18±0.07) n mM (here, n is the number of CH2) to 0.12±0.05 (p<0.01) and 0.15±0.05 (p>0.05) and (1.18±0.07) n mM (p<0.01). After 14 days differentiation, the cells shape transformed into ovoid/round morphology and many lipid droplets distributed in the cytoplasm appeared jacinth with the red-0 staining. In addition, the mRNA expression of adipocyte-specific genes, such as peroxisome proliferator-activated receptor (PPAR-r), acyl-CoA synthetase (ACS), and the enzyme lipoprotein lipase (LPL) in the MSCs were increased obviously.Conclusion:The biomarker features of MSCs were obtained by using high resolution MR spectroscopy. The 1.28ppm biomarker considered as unique for NPCs was also observed in the MSCs spectra, but it is not specific biomarker for the stem cells. MR spectroscopy is capable of monitoring the adipogenic differentiation of the MSCs, according to the metabolites change.