Diverse effects of dimethyl sulfoxide (DMSO) on the differentiation potential of human embryonic stem cells

Abstract

In vitro disease modeling using pluripotent stem cells can be a fast track screening tool for toxicological testing of candidate drug molecules. Dimethyl sulfoxide (DMSO) is one of the most commonly used solvents in drug screening. In the present investigation, we exposed 14- to 21-day-old embryoid bodies (EBs) to three different concentrations of DMSO [0.01% (low dose), 0.1% (medium dose) and 1.0% (high dose)] to identify the safest dose that could effectively be used as solvent. We found that DMSO treatment substantially altered the morphology and attachment of cells in concurrence with a significant reduction in cell viability in a dose-dependent manner. Gene expression studies revealed a selective downregulation of key markers associated with stemness (Oct-4, Sox-2, Nanog and Rex-1); ectoderm (Nestin, TuJ1, NEFH and Keratin-15); mesoderm (HAND-1, MEF-2C, GATA-4 and cardiac-actin); and endoderm (SOX-17, HNF-3β, GATA-6 and albumin), indicating an aberrant and untimely differentiation trajectory. Furthermore, immunocytochemistry, flow cytometry and histological analyses demonstrated substantial decrease in the levels of albumin and CK-18 proteins coupled with a massive reduction in the number of cells positive for PAS staining, implicating reduced deposits of glycogen. Our study advocates for the first time that DMSO exposure not only affects the phenotypic characteristics but also induces significant alteration in gene expression, protein content and functionality of the differentiated hepatic cells. Overall, our experiments warrant that hESC-based assays can provide timely alerts about the outcome of widespread applications of DMSO as drug solvent, cryoprotectant and differentiating agent.

Dimethyl sulfoxide induces chemotherapeutic resistance in the treatment of testicular embryonal carcinomas

“…In the present study, DMSO reduced the expression of stemness-related markers, such as SOX2, in the human EC cells, while also reducing DNMT3L, which is a specific marker for human EC cells. DMSO is known to induce differentiation in ES cell lines (25,26). Together with the perturbed differentiation of the human EC cells observed in response to DMSO within the present study, the findings also support a role for DMSO in modifying the stemness characteristics of EC cells and in mediating differentiation from pure EC cells to the other phenotypic components, including teratoma, choriocarcinoma and yolk sac tumors.…”

“…The present study results indicated that DMSO can induce resistance to CDDP and can perturb the differentiation status of human EC cells. DMSO is frequently used as a solvent in biological studies and as a vehicle for drug therapies (25,26). Even if DMSO is used as a vehicle for other drugs, when these agents are used in combination with CDDP, DMSO could reduce the chemotherapeutic efficacy of human EC cells.…”

Implementation and Challenges of Direct Acoustic Dosing into Cell-Based Assays

“…3 The small-volume transfers enabled by ADE mitigate the impact of the use of DMSO on biological assays. DMSO is the routine solvent for pharmaceutical small-molecule compound collections; however, DMSO has been shown to induce a range of effects on cells, including differentiation, growth arrest, and reduced cell viability, [4][5][6] and DMSO tolerance in cell-based assays is typically 0.1% to 0.5% v/v. Miniaturization of assays into 384-well and 1536-well formats is also enabled.…”

Hepatic differentiation of human embryonic stem cells in a 3D bioreactor environment

“…The stimulation with BMP4 together with FGF2 has been shown to induce mesendodermal differentiation Another small molecule used for the hepatic differentiation is dimethylsulfoxide (DMSO). It was reported to have a dose dependent effect on the expression of a large number of genes 113 . In the past DMSO has been widely used for the cryopreservation of cells, but has recently been found to also affect the undifferentiated state of hESC 114 .…”