The overall goal of the following experiment is to derive neuronal progeny directly and exclusively from pluripotent human embryonic stem cells using small molecule induction. This is achieved by adding rettino acid to undifferentiated cells maintained under defined conditions. Next, the differentiation process is continued by detaching cells in a suspension culture where they form floating cellular clusters called neuroblast.
If neuroblast are permitted to attach to a tissue culture plate in neuro differentiation media, they develop characteristics of mature neurons. Ultimately, immunofluorescence and deconvolution or confocal microscopy can be used to show the effect of rettino acid treatment in morphology and marker expression of treated human embryonic stem cells. In other techniques, only a small fraction of cells pursue a neuronal phenotype, whereas our method allows well-controlled efficient induction of pluripotent human embryonic stem cells exclusively towards a neuronal lineage By simple provision of small molecules.
There are a few steps to be careful with when repairing 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 ESL media, some of the supplements are added just before it is used.
These include BFGF, ascorbic acid, human insulin active in A and transferring. Next, make the matra gel 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 matrigel 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%Under differentiated human ES cells, they're usually slightly opaque with defined edges colonies containing piled up cells. 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 fibroblasts 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 E es cell media lacking BFGF, and then add three milliliters of fresh human ESL 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. After seeding for three days, make new media containing omic acid. 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 to each. Well Add four milliliters of fresh human ES cell media with BFGF and rettino acid. Replace the media every other day and allow rettino acid treated human ES cell colonies to grow.
After seven or eight days, the cells will have undergone morphological changes into large differentiated cells. Then proceed with making a suspension cell 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.
During this time, floating cellular clusters or neuroblast will form to further advance the neuroblast to a more mature neuronal phenotype. Pull the media containing them into a 50 milliliter conical tube. 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 NSC media containing VEGF N NT three and BDNF. Mix the cells into a suspension by pipetting, then aliquot four milliliters of the suspension per well into six. Well tissue culture plates transfer the plates to a 37 degree Celsius humidified incubator.
The neuroblast will attach to the culture plates overnight for A 3D culture at Matrigel or human laminate to the neuroblast suspension. And a 3D matrix will polymerize at 37 degrees Celsius. Replace the media every other day.
Within two weeks, extensive networks of neurite bearing neuronal cells and pigmented cells will appear rettino acid is rendered sufficient to induce human ES cells maintained in the defined culture system to transition from pluripotency exclusively to a neuro epidermal phenotype. Upon exposure of undifferentiated human E es cells to retinoic acid, all the cells within the colony underwent morphological changes into large differentiated cells that ceased to express the pluripotency associated marker OCT four. The cells also began to express various neuroectoderm associated markers such as HNK one a, P two, and TRKC.
These large differentiated cells continued to multiply and the colonies increased in size spontaneously expressing the early neuronal marker beta three tubulin. The more mature neuronal marker map two began to appear in areas of the colonies where cells had piled up. Coincidentally, with the appearance of the neuroectodermal cells, the neuronal specific transcription factor NER one implicated in dopaminergic neuronal differentiation and activation of the tyrosine hydroxylase gene translocated into the nucleus.
After detachment, the rettino acid treated human ES cells formed neuroblast in a suspension culture upon removal of basic FGF and after permitting the neuroblast to attach to a tissue culture plate, extensive networks of neurite bearing neuronal cells and pigmented cells, typical of the CNS began to appear with a drastic increase in efficiency and could be cultured over three months. These human ES cell derived neuronal cells expressed beta three tubulin compared to spontaneous multi lineage differentiation of cells without rettino acid treatments and co-expressed map two Once mastered. This technique can be used to generate neuronal progenitors in neuronal cells uniformly from pluripotent human embryonic stem cells in about four weeks if it is performed properly.
Don't forget that working with human cells can be extremely hazardous and precautions such as ensuring cell lines are free of pathogens and mycoplasma should always be taken before performing this procedure.
