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

It is a simple treatment that can be applied to any stem cell line or differentiation protocol to prime cells for differentiation. All one has to do is to add DMSO at 1-2%into the cell culture maintenance medium. The main advantage of this technique is it allows one to significantly increase the differentiation potential of pluripotent stem cells into any lineage of choice by priming cells for differentiation.

DMSO treatment increases the chance that cells will differentiate from patient-derived iPSCs, allowing them to be used for cell replacement therapy, disease modeling, and drug screening. It can give rise to the early onset of regulation that control sub-proliferation, differentiation, and specification during embryonic development. We have demonstrated that the DMSO treatment improves differentiation in nearly 50 human embryonic and induced pluripotent stem cell lines.

While other laboratories have shown that it's beneficial for stem cells of other species, such as mouse and primate. One day before the onset of differentiation, add DMSO at a concentration of 1 or 2%for 24 hours into your typical maintenance medium. 24 hours after the DMSO treatment, replace medium and proceed with your usual differentiation protocol.

Grow cells according to manuscript directions. When the cells reach an appropriate confluency, dissociate them and prepare a single cell suspension. Count the live cells with a hemocytometer or automatic cell counter, using a viability marker such as trypan blue.

In order to achieve 80-90%confluency within the 24-hour DMSO pre-treatment, plate the cells onto a coated six-well plate at a density of 500, 000 to 1 million cells per well. Incubate the cells for 24 hours at 37 degrees Celsius in a carbon dioxide incubator. Then, aspirate the media and replace it with a 1-2%DMSO solution.

Allow the cells to incubate for 24-28 hours prior to differentiation. If incubating for 48 hours, replace the media after 24 hours with fresh DMSO solution. To achieve 3D culture differentiation, grow and collect the cells in a cell suspension.

Count the live cells and plate them in an uncoated, low-attachment six-well plate in stem cell media with ROCK inhibitor. Typically, 3D human pluripotent stem cells spheres will form within 24 hours. Allow the cells to incubate for 24 hours at 37 degrees Celsius in a carbon dioxide incubator.

Prepare 1-2%DMSO in pre-warmed stem cell media according to manuscript directions. To replace the media after incubation, tilt the plate to a 30 degree to 45 degree angle and allow cell spheres to settle at the bottom of the well. Aspirate the media from the cells and gently re-suspend them in the DMSO solution.

Allow the cells to incubate for 24-48 hours at 37 degrees Celsius in a carbon dioxide incubator. Pre-treat cells with DMSO as described for 2D cultures, and prepare noggin and SB431542 stock solutions. Prepare enough 10%knockout serum replacement, or KOSR, in knockout DMEM for three to four days of media change.

Prepare ectodermal differentiation media by adding noggin and SB431542 to pre-warmed KOSR knockout DMEM as described in the manuscript. After DMSO pre-treatment, aspirate media from cells and add two milliliters of differentiation media to each well. Allow the cells to incubate for three to four days at 37 degrees Celsius in a carbon dioxide incubator, replacing media daily with fresh differentiation factors.

DMSO pre-treatment results in a transient and dose-dependent decrease in cell growth rate compared to non-treated cells. This decrease proliferation is associated with an increase in cell-to-cell contact, which is especially pronounced in the 2%DMSO-treated cells. When the cells are fixed and stained for prototypic markers of each germ layer, it can be seen that 2%DMSO pre-treatment results in increased proportion of differentiated germ layer cells.

For example, SOX17 was stained to visualize endoderm cells. In order to investigate the effect of DMSO on differentiation to central nervous system progenitor cell types, human-induced pluripotent stem cells could be differentiated into either neural progenitor cells or oligodendrocyte progenitor cells. Compared to control cells, 2%DMSO pre-treatment increases the expression of neural progenitor cell marker Pax6.

Similarly, the pre-treatment increases the proportion of cells expressing oligodendrocyte progenitor cell marker Olig2. Furthermore, initial DMSO treatment improves human embryonic stem cell-derived cell function following in-vivo transplantation. When differentiated pancreatic progenitor cells are transplanted into immunodeficient mice, their functionality can be tested in response to a glucose challenge or KCl stimulation.

Improvements in cell functionality are evident from two weeks to 16 weeks post-transplantation. The most important consideration to take into account is the cycling or doubling time of your stem cells. The length of DMSO treatment should be the same as the cycling time of the cells.

Following the initial DMSO treatment, one can understand how cell proliferation and the cell cycle are regulated in stem cells, as the treatment helps promote growth arrest in the G1 phase of the cell cycle. The differentiated cells can be used to understand disease-related mechanisms, for co-culturing with other cell types, in tumorigenicity assays, and in functional assays or transplantation studies to determine if the differentiated cells can rescue a disease phenotype.