A transient DMSO treatment increases the differentiation potential of human pluripotent stem cells through the Rb family

Abstract

The propensity for differentiation varies substantially across human pluripotent stem cell (hPSC) lines, greatly restricting the use of hPSCs for cell replacement therapy or disease modeling. Here, we investigate the underlying mechanisms and demonstrate that activation of the retinoblastoma (Rb) pathway in a transient manner is important for differentiation. In prior work, we demonstrated that pre-treating hPSCs with dimethylsulfoxide (DMSO) before directed differentiation enhanced differentiation potential across all three germ layers. Here, we show that exposure to DMSO improves the efficiency of hPSC differentiation through Rb and by repressing downstream E2F-target genes. While transient inactivation of the Rb family members (including Rb, p107, and p130) suppresses DMSO’s capacity to enhance differentiation across all germ layers, transient expression of a constitutively active (non-phosphorylatable) form of Rb increases the differentiation efficiency similar to DMSO. Inhibition of downstream targets of Rb, such as E2F signaling, also promotes differentiation of hPSCs. More generally, we demonstrate that the duration of Rb activation plays an important role in regulating differentiation capacity.

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

“…As shown in Figure 2B, 24 h of pretreatment with 2% DMSO increased the proportion of cells expressing each respective germ layer marker. This is consistent with previous reports from our lab showing increased immunoreactivity, gene expression, as well as absolute number of differentiated cells towards all germ layers in stem cells treated with DMSO 3,5 . HUES6 is an hESC line with very low propensity for differentiation across all lineages 1 , yet the DMSO treatment substantially improves its capacity to differentiate across all germ layers.…”

“…Treatment with DMSO increases the proportion of hiPSCs and hESCs in the early G1 phase of the cell cycle and activates the retinoblastoma protein (Rb) 3 , a critical regulator of cell proliferation, survival, and differentiation 4 . In more recent work, it has been found that Rb and its family members are required for the pro-differentiation effects of DMSO, such that transient inactivation of Rb suppresses the effects of DMSO, while constitutive activation of Rb in a transient manner enhances DMSO's effects 5 . Analogous to the cell cycle during embryonic development, the cell cycle of ESCs and iPSCs is characterized by an abbreviated G1 phase that promotes self-renewal 6,7,8 .…”

“…To date, DMSO pretreatment has been shown to improve the differentiation capacity to all three germ layers in over 30 control and diseasespecific human ESC and iPSC cell lines 3,5 as well as the differentiation of stem cells and other cell lines to a variety of other mature cell types in subsequent studies 10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28 (Table 1). Furthermore, DMSO treatment has been shown to be effective in enhancing differentiation of non-human primary cells 21,23 (e.g., mouse, primate, rabbit), suggesting shared mechanisms across species.…”

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

“…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 .…”

“…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.…”