Marmoset induced pluripotent stem cells: Robust neural differentiation following pretreatment with dimethyl sulfoxide

Abstract

The marmoset is an important nonhuman primate model for regenerative medicine. For experimental autologous cell therapy based on induced pluripotent (iPS) cells in the marmoset, cells must be able to undergo robust and reliable directed differentiation that will not require customization for each specific iPS cell clone. When marmoset iPS cells were aggregated in a hanging drop format for 3 days, followed by exposure to dual SMAD inhibitors and retinoic acid in monolayer culture for 3 days, we found substantial variability in the response of different iPS cell clones. However, when clones were pretreated with 0.05% – 2% dimethyl sulfoxide (DMSO) for 24 hours, all clones showed a very similar maximal response to the directed differentiation scheme. Peak responses were observed at 0.5% DMSO in two clones and at 1% DMSO in a third clone. When patterns of gene expression were examined by microarray analysis, hierarchical clustering showed very similar responses in all 3 clones when they were pretreated with optimal DMSO concentrations. The change in phenotype following exposure to DMSO and the 6 day hanging drop/monolayer treatment was confirmed by immunocytochemistry. Analysis of DNAcontent in DMSO-exposed cells indicated that it is unlikely that DMSO acts by causing cells to exit from the cell cycle. This approach should be generally valuable in the directed neural differentiation of pluripotent cells for experimental cell therapy.

Overcoming barriers to reprogramming and differentiation in nonhuman primate induced pluripotent stem cells

“…The following day the medium was aspirated, and the cells were disassociated with Accutase. Cells were collected, centrifuged and suspended at a concentration of 3000 cells per 30 µL in the differentiation medium previously described (Qiu et al, 2015). The lid of a 96-well plate was used to create numerous 30 µL droplets, each containing 3000 cells.…”

“…After this time the cells were collected by flushing the lids with DMEM/F12 into a 15 mL conical tube and allowed to settle under gravity. The medium was then aspirated, and the embryoid bodies were resuspended in 2 mL of a second differentiation medium, also as previously described (Qiu et al, 2015), and plated in a nonadherent 35 mm dish. Cells were maintained in the same medium for 72 h. Following this period, cells were harvested for preparation of RNA.…”

“…Cells of each clone were subjected to a directed neural differentiation protocol previously employed in this lab (Qiu et al, 2015). After 7 days the differentiated cells were assayed for the expression levels of NCAD, POU3F1, SOX1, NKX6.6 and OLIG2 by qPCR.…”

“…They have a relatively short life span for a primate, and they are more easily maintained under laboratory conditions than other NHPs used in biomedical research (Mattison and Vaughan, 2016). Our lab has expertise in the generation of iPS cells from marmosets (Wu et al, 2010) and experience in directed neural differentiation protocols optimized for marmoset iPS cells Qiu et al, 2015) (Fig. 1a).…”

“…In both cases, cells derived from aged marmosets and cells derived by different methods from chimpanzee cells, we encountered barriers to appropriate differentiation that could be overcome in most cases by prior treatment with dimethyl sulfoxide (DMSO). DMSO pretreatment is a technique that was originally described to optimize the differentiation of human pluripotent cells to insulin-secreting cells (Chetty et al, 2013) and was found by our group to be valuable in eliminating clonal variation in differentiation potential for NHP cells (Qiu et al, 2015).…”