Transient Treatment of Human Pluripotent Stem Cells with DMSO to Promote Differentiation

Abstract

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and iPSCs) across various lineages remain. Numerous differentiation protocols have been developed, yet variability across cell lines and low rates of differentiation impart challenges in successfully implementing these protocols. Described here is an easy and inexpensive means to enhance the differentiation capacity of PSCs. It has been previously shown that treatment of stem cells with a low concentration of dimethyl sulfoxide (DMSO) significantly increases the propensity of a variety of PSCs to differentiate to different cell types following directed differentiation. This technique has now been shown to be effective across different species (e.g., mouse, primate, and human) into multiple lineages, ranging from neurons and cortical spheroids to smooth muscle cells and hepatocytes. The DMSO pretreatment improves PSC differentiation by regulating the cell cycle and priming stem cells to be more responsive to differentiation signals. Provided here is the detailed methodology for using this simple tool as a reproducible and widely applicable means to more efficiently differentiate PSCs to any lineage of choice.

Brief Report: Cell Cycle Dynamics of Human Pluripotent Stem Cells Primed for Differentiation

“…A transient 24 hours DMSO treatment also does not affect cell toxicity as cell viability is comparable in untreated control and DMSO‐treated hPSCs prior to differentiation (Supporting Information Fig. S6D, S6E), consistent with prior reports .…”

“…In a prior study, we demonstrated that transiently treating hPSCs with dimethylsulfoxide (DMSO) for 24 hours prior to directed differentiation significantly increases the propensity for differentiation across all germ layers . This technique is now used by multiple laboratories to improve differentiation across species (including mouse, rabbit, primate, and human) into more than a dozen lineages, ranging from neurons and cortical spheroids to smooth muscle cells to hepatocytes . Although the DMSO treatment activates the retinoblastoma protein (Rb) and increases the percentage of hPSCs in the G1 phase of the cell cycle , it remains unknown whether the DMSO treatment simply enriches cells in G1 or whether there are intrinsic changes to the cell cycle following the DMSO treatment that may potentiate differentiation.…”

Generation and transplantation of hepatocytes‐like cells using human origin hepatogenic serum for acute liver injury treatment

“…Group 5S + D showed a marked change in morphology of the HLC as compared to the other groups (Figure 4C). DMSO is well known to induce hepatogenesis in various types of stem cells, that is, ESC, 16 iPSC, 19,20 and MSC. 17 Expression of hepatocytes-specific protein markers are important for assessing the quality control of derivation strategies employed for differentiation to a terminal cell type using stem cells.…”

Effects of DMSO on the Pluripotency of Cultured Mouse Embryonic Stem Cells (mESCs)

“…According to the literature, DMSO is commonly used in the differentiation protocol of several cancer cell lines [ 30 , 31 , 33 ] and embryonic stem cell lines [ 8 , 13 , 16 – 18 ] in both mouse and human. In fact, recently several protocols have been established where DMSO pretreatment of human pluripotent cells is the key step to enhance efficiency of the differentiation protocol [ 19 , 34 ]. Yet, our analysis showed that exposure to much smaller amounts of DMSO for 48 hours did not affect pluripotency status or differentiation potential.…”