Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

Human embryonic stem cells can be induced to differentiate into neural progenitor cells and subsequently into neuron astrocyte and oligodendrocyte. However, type culture method for human embryonic stem cells and their differentiation into neural progenitor cells are very inefficient and open in vivo co-culture system Here we present an improved and robust culture system that is easily scalable using high-density single cell type culture with human embryonic stem cells. Single cell type culture of human embryonic stem cells provides a rapid and efficient system to study the molecular mechanisms that regulate the multi-step differentiation along various and distinct differentiated lineages, including neural progenitor cells and their subsequent differentiation into additional neural lineages.

Begin by preparing human embryonic stem cell qualified basement membrane matrix coated plates. Slowly thaw the basement membrane matrix solution at four degrees Celsius for at least two to three hours or overnight. When thawed, dilute the matrix in cold DMEM/F-12 to 2%mix well, and coat each well of a six-well plate with one milliliter of the diluted solution.

Incubate the coated plates at room temperature for at least three hours or at four degrees Celsius overnight. To passage the feeder-free cultures of colony type H9 human embryonic stem cells grown on basement membrane matrix, aspirate the medium from the wells and wash once with one milliliter of DPBS. Add one milliliter of Dispase solution to each well and incubate the plate at 37 degrees Celsius for 20 minutes.

Remove the Dispase and gently wash the cells once with two milliliters of DMEM/F-12. After the wash, remove the medium and add two milliliters of DMEM/F-12 to each well. Gently detach the colonies by pipetting up and down and transfer them to a 15 milliliter tube.

Centrifuge the tube at 370 times g for two minutes. Aspirate the medium. Then dissociate the cell pellets into single cells by adding two milliliters of cell detachment solution and incubating them at 37 degrees Celsius for 10 minutes.

Repeat the centrifugation and remove the detachment solution. Resuspend the cells in mTeSR-1 human ESC medium by gently pipetting up and down. To adapt the colony type human embryonic stem cells to a single cell type culture, plate approximately 1.5 to two million cells into each well of the basement membrane matrix coated plate in two milliliters of mTeSR-1 containing 10 micromolar ROCK inhibitor.

After 24 hours, replace the medium with fresh mTeSR-1 without ROCK inhibitor. Allow the cells to grow as a single cell type for three days changing the medium daily. On the fourth day, dissociate the cells in detachment solution and re-plate them as previously described.

After resuspending and incubating the cells according to the manuscript directions, transfer the small embryonic bodies to a 15 milliliter tube, let them settle to the bottom, and gently remove the medium with a pipette. Transfer them into EB medium and allow them to expand in low attachment dishes for seven days. To induce neural progenitor cell or NPC differentiation, dissociate the single cell type human embryonic stem cells with one milliliter of detachment solution and incubate them at 37 degrees Celsius for 10 minutes.

Centrifuge the cells for two minutes at 370 times g. Remove the detachment solution supernatant. Then resuspend the cells in DMEM/F-12.

Plate the cells on a basement membrane matrix coated six-well plate at a density of two times 10 to the fifth cells per well in two milliliters of mTeSR-1 with 10 micromolar ROCK inhibitor. After 24 hours, replace the culture medium with neural induction medium supplemented with one micromolar Dorsomorphin and five micromolar SB431542. Change the medium every other day during the first four days of neural induction, then everyday until confluence is reached at day seven.

After seven days of neural induction, dissociate the cells by adding one milliliter of detachment solution and incubating them for 10 minutes at 37 degrees Celsius. Centrifuge the cells at 370 times g and remove the detachment solution supernatant. Add one milliliter of NPC expansion medium and gently pipette the cells up and down to resuspend.

Then plate one times 10 to the fifth cells per well in two milliliters of NPC expansion medium on the basement membrane matrix coated six-well plates. Passage the cells as necessary when cultures reach 90%confluency adding 10 micromolar ROCK inhibitor during the first three to four passages. Change the medium every other day during the expansion.

To prepare PLO laminin-coated plates, dilute the PLO stock solution in PBS or water to a final concentration of 10 micrograms per milliliter, mix well, then coat each well of a six-well plate with one milliliter of the solution. Incubate the plates for at least two hours at 37 degrees Celsius or overnight at four degrees Celsius. Next, wash each well with PBS and immediately after coat each well with one milliliter of laminin solution prepared according to the manuscript directions.

Keep the plates at four degrees Celsius for up to one week. To differentiate NPCs into dopaminergic neurons, plate the cells in PLO laminin-coated dishes in NPC expansion medium at a density of approximately 50%After 24 hours, change the medium to DA1 and change the medium every other day thereafter. To passage cultures when they reach confluency, dissociate the cells with one milliliter of detachment solution and incubate them for 10 minutes at 37 degrees Celsius.

Then centrifuge them at 370 times g and remove the detachment solution supernatant. Resuspend the cells in one milliliter of DA1 medium and plate one times 10 to the fifth cells in two milliliters of medium on the PLO laminin-coated six-well plates. To differentiate the NPCs into astrocytes, plate the cells on the PLO laminin plates and NPC expansion medium at a density of 50%change the medium to astrocyte medium after 24 hours, and passage the cells as previously described.

When colony type human embryonic stem cells were adapted to the single cell type culture, it was found that the cells were able to be maintained at high density, then easily and efficiently subcultured when confluency was reached. These cells retained the cell cycle characteristics typical of colony type human embryonic stem cells such as a short G1 phase and a high proportion of cells in S phase. Quantitative PCR analysis also confirmed that they express ESC markers at levels comparable to those of colony type cells.

Moreover, it was shown that single cell human embryonic stem cells were able to form embryoid bodies containing cells from all three germ layers:endoderm, mesoderm, and ectoderm. It was then demonstrated that single cell type human embryonic stem cells efficiently differentiated into NPCs as indicated by the loss of typical human embryonic stem cell morphology and appearance of NPC morphology. Differentiation was supported by the increased expression of signature NPC markers and confirmed by immunostaining and FACS analysis.

The same analysis also showed that more than 90%of the cells stained positive for SOX1, PAX6, and NCAM proteins. Furthermore, the derived NPCs were able to differentiate into dopaminergic neurons and astrocytes as indicated by the appearance of characteristic morphologies and expression of lineage-specific markers. When attempting this procedure, it is essential to plate the human embryonic stem cells at high density in order to maintain a healthy, undifferentiated human embryonic stem cell culture.

This protocol provide a platform for simple, robust, and scalable production of progenitor and differentiated cell that will be scalable for basic study, drug screen, and application in neurodegenerative medicine.