The overall goal of the following experiment is to differentiate human pluripotent stem cells or H PSCs into neurons using a multi titer plate format. This is achieved by harvesting and plating a single cell suspension of H PSCs into V bottom 96. Well plates to form embryo bodies or EBS overnight.
Next, the EBS are transferred into U-shaped ultra-low attachment plates containing differentiation medium to induce neuroectoderm formation. Subsequently, the EBS are plated onto matrigel coated dishes to promote the outgrowth of neurons. Results can be obtained that show the presence of typical neuronal markers around day eight by immunofluorescence.
Staining Protocols for differentiating human pre potent stem cells into neurons are often complicated and tedious. Somewhat accidentally. We came across a very simple procedure for making neurons from human pre potent stem cells.
From a technical point of view, the main advantage of this technique of existing methods is that it's easy to carry out, rapid and cost efficient. At the same time, presenting the procedure will be Dr MI Z from the lab. Prepare EB formation medium and differentiation medium as outlined in the written protocol.
Grow h PSCs under feeder free conditions such as a meth conditioned medium on matrigel coated plates, or using chemically defined culture systems under sub confluent actively growing conditions. Using a stereo microscope with a sterile plastic pipette tip mechanically removes spontaneously differentiated colonies. Wash remaining undifferentiated colonies with PBS and use Accutane containing one x rock inhibitor to digest to single cells.
Pellet the single cells by centrifugation at 200 G for two minutes and Resus suspend in a small volume of EB formation medium. Use a hemo cytometer to determine the cell titer. Add additional cells to the remaining EB formation medium to arrive at a titer between 40, 000 and 80, 000 cells per milliliter.
Using a multi-channel pipette transfer 100 microliters per well to a 96 V bottom plate, resulting in 4, 000 to 8, 000 cells per.Well. Spin the plate in a swing up plate centrifuge at 400 G for one minute to collect all cells at the bottom of the wells. Transfer the cells to an incubator and allow EBS to form overnight the next day under the stereo microscope.
Collect EBS and transfer in a small volume into a 3.5 centimeter bacterial dish containing two milliliters of differentiation.Medium. Wash the cells by gently rotating the dish. Add 100 microliters of differentiation medium to each well of an ultra low attachment you.
Bottom 96 well plate under a stereo microscope. Transfer EBS in small volumes from the washing dish into the 96 U wells. Incubate the EBS for four days to allow neuroectoderm formation on the fourth day.
Wash the cells in differentiation medium and seed them one by one into the wells of a matrigel coated multi titer plate, such as a 12 well plate. The cells should be equally distributed within the wells to allow undisturbed outgrowth of neurons. Allow the EBS to loosely attach to the matrigel for approximately 10 minutes at room temperature.
Then carefully transfer the plate to the incubator. Neurons will grow out from the plated EBS in a radial manner within the next four days. Previously we showed that in line with many others, EBS from H PSCs could be generated simply by replating, aggregates upon routine splitting of H HPCs into low attachment dishes.
This usually results in a wide distribution of cell sizes. Another problem is that EBS maintained in suspension tend to aggregate with one another leading to even higher size heterogeneity to circumvent this single H PSCs were pleaded in low attachment wells, which resulted in no EB formation and cell death. However, combining the cell survival molecule, Y 2 7 6 3 2 with polyvinyl alcohol, which has been shown to enhance EB formation from HSCs, allowed EBS to form from single cell HESC suspensions overnight.
Conveniently, EB sizes in this multi titer plate format could be tightly controlled by seating different numbers of cells per well. In contrast to using conventional techniques, after transferring EBS to ultra-low attachment 96 U plates, neuroectoderm formation was induced using a small molecule cocktail consisting of F-G-F-E-R-K-T-G, TGF beta SMA two, and BP SMA one inhibitors, which then allowed the cells to be plated on matrigel to give rise to neurons. Initiating EB formation from around 8, 000 cells yielded the best neuronal differentiation capacity, which is characterized by pronounced neuronal outgrowths from the plated ebs.
Reduced necrotic EB centers after plating and high expression of sensory and pan neuronal markers such as B rrn three A and beta three tubulin. The overall neuronal differentiation efficiencies appear to be comparable to the original procedure, which is applicable to both HSCs and HI PSCs thus far. Three independent HPSC lines, HES as well as HIPS cells have successfully been tested in the multi-well procedure, whereas cell line dependent variability and differentiation efficiencies can generally not be ruled out.
This figure shows dissociated single day four neuroectodermal cells that were plated out as single cells and successfully differentiated at approximately 60%efficiency Handling EBS in the multi titter platform requires a bit of training in the beginning, but after some time, you will see it's quite easy.
