The overall goal of the following experiment is to derive cardiac progeny directly and exclusively from pluripotent human embryonic stem cells using small molecule induction. This is achieved by adding nicotinamide to undifferentiated human ES cells maintained under defined conditions. Next, cardiac differentiating cells are detached to form cardio blasts, floating cellular clusters in a suspension culture.
Once formed, cardio blasts are attached to cardiac differentiation media where they continue to develop into beating cardiomyocytes. Ultimately, results are assessed by immunofluorescence with deconvolution or confocal microscopy and by electrophysiological recordings. The main advantage of this technique is that it allows efficient induction of pluripotent human embryonic stem cells exclusively towards a cardiac lineage by simple provision of small molecules.
Whereas in existing methods of multi lineage differentiation, only a small fraction of cells pursue a cardiac phenotype. There are a few steps to be careful with when preparing stock solutions. Take care to slow thaw matrigel and human laminin from minus 80 degrees Celsius to four degrees Celsius overnight store media at four degrees Celsius and always warm up the media to 37 degrees Celsius before use.
Also, when preparing human E es cell media, some of the supplements are added just before it is used. These include BFGF, ascorbic acid, human insulin active in A and transfer in. Next, make the matrigel or human laminin working solutions immediately before plate coating.
Incubate the plates at 37 degrees SIUs in 5%carbon dioxide After the gelatin pre-coated tissue culture plates are prepared. The gelatin can be replaced by the matra gel or laminin working solution. Coat these plates by incubating them overnight at four degrees Celsius.
Begin by allowing human ES cell colonies to grow for five to seven days and then prepare to split some of them Select colonies with more than 75%undifferentiated human ES cells. They're usually slightly opaque with defined edges colonies containing piled upsells. An indication of the beginning of differentiation usually appear to be white colonies containing mostly differentiated cells usually appear to be clear.
Do not select white or clear colonies. Hold the plates up to the light and use a marker to outline the slightly opaque colonies on the bottom of the plates along the outlines. Use the edge of a sterile P two pipette tip to detach the surrounding fibroblasts layer from the human ES cell colony.
Then remove the surrounding fibroblast cells and also remove all the differentiated parts of the colony. Now aspirate away the old media containing the detached differentiated cells. Next in each well wash the undifferentiated cells once with human ES cell media lacking BFGF, and then add three milliliters of fresh human ES cell media containing B-F-G-F-B-F-G-F is added to the fresh media immediately before use.
Now use a sterile P two pipette tip to cut the colonies into small pieces and detach them from the plate. Pull the detached colony pieces in their media into a 50 milliliter conical tube. Pull an additional one milliliter media rinse per well.
The cells are now ready for further differentiation using small molecules. Begin by warming freshly coated plates to 37 degrees Celsius. Aspirate the coating solution from the plates and add a four milliliter aliquot of media containing colony pieces into each Well then gently transfer the plates to an incubator without shaking them.
Do not disturb the plates so they are allowed to seed. Then remove most of the old media, leaving just enough media so that the cells remain submerged. The cells should never be allowed to dry out.
Mix fresh human ESL media containing basic FGF and nicotinamide and formula milliliters of this media to each well. The media can be stored for up to two weeks at four degrees Celsius, during which it can be used to refresh the cell culture every other day to grow the colonies. After eight to 10 days, the cells will have undergone morphology changes to large differentiated cells multiplied and piled up.
Then proceed with a suspension culture to make a suspension culture begin by detaching ESL colonies from cells that have spontaneously differentiated and migrated out to form a fibroblast layer. Use the aforementioned technique with a sterile pipette tip, then aspirate away the old media containing floating detached fibroblast cells. Wash the cells once with HESC media and re suspend them in three milliliters of HESC media now using a sterile P two pipette tip.
Cut the colonies into small pieces and detach them from the plate. Pull the media with detached colonies into a single 50 milliliter conical tube. Rinse the wells with one milliliter of media and add the rinse to the pool.
Then aliquot four milliliters of the pooled cells into each well of a six well ultra-low attachment plate for four to five days. Keep the plate in an incubator to allow floating cellular clusters or cardio blasts to form to advance the cardio blasts to the beating cardiomyocyte phenotype. Pull them together in a 50 milliliter conical tube with their media.
Then centrifuge the cells at 1, 400 RPM for five minutes. Aspirate, weigh as much of the old media as possible and add an equal amount of fresh cardiac differentiation. Media containing 10 millimolar nicotinamide.
Mix the floating cardio blasts with pipetting, then aliquot four milliliters of the cardio blasts suspension into each well of six well tissue culture plates. Transfer the plates to a 37 degrees Celsius humidified incubator to allow the cardio blasts to attach to the tissue culture plates overnight. Every other day in culture.
Replace the cardiac differentiation media containing 10 mini molin nicotinamide on day eight. Switch to cardiac differentiation. Media without nicotinamide continue to replace the media every other day.
Within two weeks after the withdrawal of nicotinamide beating cardiomyocytes will begin to appear and increase in number nicotinamide is rendered sufficient to induce human ES cells maintained in the defined culture system to transition from pluripotency exclusively to a cardio mesodermal phenotype. Upon exposure of undifferentiated human ES cells to nicotinamide all the cells within the colony underwent morphological changes into large differentiated cells that downregulated the expression of OCT four. The cells also began to express the cardiac specific transcription factor, NK X 2.5, and also express alpha actinin.
Both characteristics are consistent with the cardio mesoderm phenotype. These differentiated cells continue to multiply and increase in size in areas of the colonies where cells had piled up. NKX 2.5 expression became more intense After detachment, the nicotinamide treated human ES cells formed cardio blasts in a suspension culture.
After permitting the cardio blasts to attach cardio blasts were treated with nicotinamide for one week, one to two weeks after withdrawal from nicotinamide beating cardiomyocytes began to appear with a drastic increase in efficiency. They appeared as cellular clusters that pulsed with rhythmic contractions, spontaneous multi lineage differentiation of cells that did not undergo IDE treatment did not express these characteristics. Cells within the beating cardiomyocyte clusters expressed markers characteristic of cardiomyocytes, including NK X 2.5 and alpha actinin.
The contractions of the beating cardiomyocytes were confirmed by electrical profiles to be strong, rhythmic while coordinated and well entrained, they displayed regular impulses reminiscent of the P-Q-R-S-T complexes, seen from body surface electrodes in clinical electrocardiograms. Furthermore, the cardiomyocytes could retain their strong contractility for over three months Once mastered. This technique can be used to generate cardiac precursors and beating cardiomyocytes uniformly from pluripotent human embryonic stem cells in about five weeks if it is performed properly.
