ヒト多能性幹細胞のin vitro分化における絨毛細胞へ

The overall goal of this protocol is to differentiate human pluripotent stem cells into human trophoblastic cells using bone morphogenic 4 and inhibitors of the active and nodal signaling pathways to study the early events of human placental formation and function. This method can help answer key questions in the study of trophoblastic cell functions and how these cells are formed. The main advantage of this technique is that it can produce abundant trophoblastic cells for a wide variety of applications including genetics and allow cell function assays.

To begin the experiment, thaw an aliquot of extracellular matrix on ice for three to four hours. Using ice cold tips, one cold 50 milliliter conical tube and Dulbecco's modified equal medium, or DMEM F12 medium, transfer two milligrams of extracellular matrix into 24 milliliters of ice cold DMEM F12. Immediately transfer the 50 milliliter conical tube to ice and store it at four degrees Celsius.

To coat tissue culture plates, add the one milliliter of diluted extracellular matrix per well of a six-well plate. Swirl the plate to coat the wells and incubate the plate at room temperature for at least one hour. Aspirate the extracellular matrix from the new plate and add mouse embryonic fibroblast-conditioned medium or MEFCM containing bFGF.

Use a pipette to transfer the scraped cellular clamps from the MEF plate and aliquot them into the extracellular matrix-coated plate. Then incubate the plate. Scrape off differentiated cells with a Pasteur pipette before replacing the MEFCM with bFGF medium daily using two milliliters of medium for one well.

When colonies appear big and bright under the microscope, passage hESCs every six to seven days. When the feeder-free cells are ready to passage, remove the medium and wash the cells by adding one milliliter of PBS using a pipette. Aspirate the PBS and replace it with 0.5 milligrams per milliliter of pre-warmed dispase with a pipette.

Incubate the cells at 37 degrees Celsius for five minutes. After incubation, wash the cells with PBS by adding two milliliters of PBS per well and aspirate the PBS. Add one milliliter of CM per well and manually scrape the cells into small clumps using the tip of a five milliliter pipette.

Next, plate the suspended cell clumps into new extracellular matrix-coated plates. Prepare the differentiation medium. Use CM without bFGF and add fresh inhibitors prior to use.

Use feeder-free hESCs growing on extracellular matrix-coated plates for one passage cultured inside an incubator set at 5%oxygen. After the first passage onto the extracellular matrix-coated plates, let the cells attach for 24 hours. The next day, initiate differentiation by removing the CM containing bFGF and replacing it with CM containing BMP4/A/P with two milliliters per well in a six-well plate.

Continue culturing the cells using 4%oxygen levels. Replace the CM containing BMP4/A/P every two days by aspirating the old medium and adding new medium containing BMP4/A/P. Collect cells at desired time points.

Culture hESCs for two passages on extracellular matrix after transferring them from the MEFs with hESC medium containing bFGF. High cellular confluency is important so split the hESCs one-to-one onto a new plate which will ensure at least 50%confluency the next day. Incubate the cells overnight in the low oxygen incubator.

The next day, replace the medium with two milliliters per well of differentiation medium. Place the cells in a low oxygen incubator overnight. Before transfection, gelatin coat a dish adding 0.1%gelatin to a dish.

Swirl the dish to coat the plate, then leave the plate at room temp. After 20 minutes, remove the gelatin. Differentiate the cells until the desired time point.

The day before transfection, trypsinize the cells using 0.05%trypsin for five minutes. Plate the cells to the desired confluency onto the gelatin-coated plate. Add CM containing BMP4/A/P lacking bFGF and incubate the culture overnight.

The next day, add fresh differentiation medium to the cells. Transfect siRNAs using an siRNA transfection reagent. For plasma DNA, use an appropriate lipofectamine reagent.

Follow the product protocol as described for each transfection reagent. Transfer the siRNA lipofectamine complexes to each well or plate of cells and mix gently. Then culture the cells overnight in a low oxygen incubator.

The next day, replace the media with fresh differentiation media. Harvest the cells at the desired time points to check the gene disruption efficiency using quantitative RT-PCR. Finally, to harvest cells, add one milliliter of 0.05%trypsin per well and incubate the cells.

Add five milliliters of MEF medium per well to neutralize the trypsin. Spin down the cells at 200 times g for five minutes and use the cell pellet for RNA isolation. Using this technique, morphological changes are clearly seen.

Cells on differentiation day one are larger than the cells from day zero. As differentiation proceeds, the cells divide and expand out from the center of the colony, growing in an outward direction. The individual colonies merge together by differentiation day five with cells growing on top of each other.

QRT-PCR analysis reveals that the relative expression levels of the pluripotent C-marker NANOG are reduced by 75%after one day of differentiation when cells are cultured with BMP4 and reduced by 90%in the presence of BMP4/A/P. By differentiation day two, the levels of NANOG are very low for cells cultured in BMP4 alone or in BMP4/A/P compared to those in hESC CM medium containing beta FGF. Transfections performed using a GFP plasmid and introduced into BMP4/A/P treated cells on differentiation day one and day three obtained 30 to 50%transfection efficiency at 24 hours post-transfection.

After watching this video, you should have a good understanding of how to differentiate human pluripotent cells into trophoblastic cells in vitro and how to introduce DNA constructs for manipulating gene expression.