Mycophenolic Acid Inhibits the Proliferation and Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells by Guanosine Depletion.
Authors: Cao Weijie , Liu Lizhen , Lai Xiaoyu , Yu Xiaohong , Huang He 
Published: October 2019

Abstract
Abstract 1454
Poster Board I-477

Introduction
Mycophenolate mofetil is now widely used in transplantation as a potent immunosuppressant, whose active metabolite is mycophenolic acid (MPA). MPA inhibits de novo purine biosynthesis by reversible, noncompetitive inhibition of inosine monophosphate dehydrogenase (IMPDH). The inhibition of IMPDH in lymphocytes reduces intracellular guanine nucleotide pools, thus arrests lymphocytes proliferation. Recently investigators reported the antiproliferative effects of MPA on fibroblasts, smooth muscle cells and endothelial cells, but there is no reports of the effects of MPA on human bone marrow-derived mesenchymal stem cells (MSCs). Here we examined the effects of MPA on the proliferation and differentiation of human bone marrow-derived mesenchymal stem cells.

Methods
Bone marrow aspirates were obtained from healthy volunteers after informed consent, and MSCs were expanded from bone marrow mononuclear cells by discarding non-adherent cells. For proliferation and survival assays, MSCs were treated with MPA at the concentration of 1μM, 10μM, 50μM, and 100μM. Cell proliferation was analyzed using CCK-8 method (Dojindo). Cell viability was assessed by trypan blue exclusion. Apoptosis was detected by PI/Annexin V assay kit (Invitrogen). To assess the effects of MPA on MSCs differentiation, osteogenic differentiation and adipogenic differentiation were induced in the presence of MPA. For the detection of osteogenic differentiation, the deposited minerals was stained with silver by the method of von Kossa and Ca2+ contents was quantified with calcium colorimetric assay kit (Biovision). Adipogenic differentiation was analyzed by Oil Red O staining and Oil Red O staining extraction.

Results
In the range of 1μM to 100μM, MPA caused a significant subdued proliferation rate of MSCs in a concentration- and time-dependent manner. After 7d of incubation with MPA at the concentration of 1μM, 10μM, 50μM, and 100μM, the proliferation rate was reduced to 65.33±11.03%, 24±3.74%, 15.33±4.03%, and 15.33±6.94% respectively (P<0.01). Adding guanosine (100μM) to the culture restored the proliferation rate (P<0.01) indicating that MPA exerted antiproliferative effects by guanosine depletion. Trypan blue staining showed that there was no statistically significant difference in the ratio of living cells between MPA treated cells and the control group (P>0.05), and PI/Annexin V staining showed no apoptosis induce by MPA. Von Kossa stainnging indicated that treatment with MPA reduced Ca2+ deposition during osteogenic differentiation of MSCs, and Ca2+ quantification further confirmed that MPA inhibited osteogenic differentiation in a concentration-dependent manner. Ca2+ quantification was 78.43±12.79 μg/well and 22.8±6.58 μg/well respectively at the concentration of 10μM and 100μM of MPA, which were significantly lower than the control group(118.33±12.50ug/well, P<0.05). Oil Red O staining and Quantification of lipid contents showed that MPA had no effect on lipid production during adipogenic differentiation.

Conclusion
Our study demonstrated that MPA inhibited the proliferation of MSCs by guanosine depletion, and also inhibited the osteogenic differentiation in a concentration-dependent manner. However, MPA had no impact on adipogenic differentiation in vitro.

Disclosures
No relevant conflicts of interest to declare.