Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions

This protocol allows the derivation of sympathetic neurons from human pluripotent stem cells. These cells will enable a scientist to study human disorders that affect the sympathetic nervous system. This technique allows the derivation of sympathetic neurons in divided feeder-free meeting conditions at a high scalable efficiency.

It results in electrically active neurons in 20 days. This technique can be applied to diseases caused by a dysfunctional sympathetic nervous system, it also can be used for disease modeling, regenerative medicine, and drug screening. These cells could be used in vitro to innovate any tissue within a co-culture system, for example, for the study of cardiac tissue regulation.

When the human pluripotent stem cell culture reaches 80 to 90%confluency, big colonies with smooth bright edges should be observed. For splitting, wash the culture with PBS before treating the cells with four milliliters of 0.25 molar EDTA at 37 degrees Celsius and 5%carbon dioxide. After two minutes, flush the plate with 10 milliliters of E8 medium and transfer the detached cell suspension to a 15 milliliter conical tube.

Then transfer 500 microliters of cells into a 9.5 milliliter aliquot of fresh E8 medium. And plate the stem cells onto new vitronectin coated 100 millimeter dishes. One day before differentiation induction, coat the appropriate number of six well plates with two milliliters of basement membrane matrix per well.

And store the plates at four degrees Celsius overnight. The next morning, warm the plates to room temperature and wash the ready to split human pluripotent stem cell culture two times with fresh PBS per wash. After the second wash, treat the cells with seven milliliters of 0.5 millimolar EDTA for 15 minutes in the cell culture incubator before aspirating the floating human pluripotent stem cells and transferring the harvested cells into a 50 milliliter tube.

Add an equal volume of PBS to the tube to dilute the EDTA and collect the cells by centrifugation. Resuspend the pellet in one milliliter of day 01 differentiation medium before adding an appropriate volume of medium for accounting. Dilute the cells to a 1.25 times 10 to the fifth cells per square centimeter concentration into milliliters of differentiation medium per well.

Aspirate all of the basement membrane matrix solution from each well of the previously prepared to six well plate. Then seed two milliliters of cells into each well and place the plate in the cell culture incubator. The next morning, feed the cells with three milliliters of day 01 differentiation medium per well.

On day two of differentiation, feed the cells with three milliliters of day two to 10 differentiation medium per well, then feed the cells every other day until day 10. To aggregate the neural crest cells into spheroids, on day 10, wash the cells with PBS. And add two milliliters of dissociation medium to each well.

After 20 minutes in the cell culture incubator, transfer the floating cells into a 50 milliliter conical tube and bring the volume of suspension in the tube to 50 milliliters with fresh PBS. Collect the cells by centrifugation. And resuspend the pellet in a sufficient volume of day 10 to 14 spheroid medium for counting.

After counting, dilute the cells to a five times 10 to the fifth cells per 500 microliters concentration using day 10 to 14 spheroid medium. And plate 500 microliters of cells into each well of an ultra low attachment 24-well plate. Then return the cells to the cell culture incubator.

To induce a minimal spheroid culture, on day 11 of culture, feed the neural crest spheroids with an additional 500 microliters of day 10 to 14 spheroid medium per well. On day 12, tilt the plate to accumulate the spheroids on one side of the plate. And carefully aspirate as much medium as possible from each well without aspirating spheroids.

Then feed the spheroids with one milliliter of day 10 to 14 spheroid medium per well. To induce an expanded spheroid culture, on day 15, feed the spheroids with 1.5 milliliters of day 10 to 14 spheroid medium supplemented with 0.5 micromolar retinoic acid per well. Then return the spheroids to the cell culture incubator.

For sympathetic neuron differentiation, on day 14 or 28, tilt the plate to accumulate the spheroids on one side of the plate and carefully aspirate as much medium as possible. Feed the cells with one milliliter of sympathetic neuron medium. To break up big spheroid aggregates into smaller spheroids, add no more than one milliliter of medium per well and pipette the spheroids five to 10 times before splitting.

And remove the excess laminin fibronectin from previously prepared 24-well plates. Split the plate one milliliter of spheroids from each well on the 24-wells spheroid culture plate between four separate wells of the new coated 24-well plate. Add 250 microliters of fresh sympathetic neuron medium to each well.

And place the plate in the cell culture incubator. The next morning, replace the medium in each well with one milliliter of sympathetic neuron medium supplemented with 0.125 micromolar retinoic acid and return the plate to the cell culture incubator. After day 35, feed the neurons by carefully replacing only half of the existing medium in each well with fresh medium.

Before differentiation, the human pluripotent stem cell colonies should be round with shiny, smooth edges and little to no differentiation. The cells express the neural crest marker Sox10 from days four through 10. Neural crest cells emerge in dense darkened ridges that are visible from day six on.

These ridges also express Sox10. At the neural crest cells stage, Sox10 correlates with the cell surface marker CD49D, which can be used to sort the neural crest cells. Positive sorted and unsorted populations produced neural crest spheroids in a similar fashion.

While negative sorted cells do not aggregate properly, do not produce round, smooth healthy looking spheroids and die within three to four days. Furthermore, when neural crest spheroids are compared at day 14, the quantitative reverse transcriptase PCR for neural crest and sympathetic nerve progenitor markers, significant differences between the sorted and unsorted cells cannot be detected. On day 20 or 35, the respective minimal or extended protocols, neuroids can be observed growing in a radial pattern from the attached spheroids.

Markers related to norepinephrine synthesis and transportation are expressed at this stage, as well as Hox6 through nine trunk neural crest genes. Electrophysiological recording also detects neural activity from day 20 on. Such activity can be enhanced or suppressed by drugs that target autoreceptors of sympathetic neurons adjusting the functionality of differentiated neurons.

The human proponent themselves must be healthy starting on day zero of differentiation, otherwise, the experiment will likely fail. Pharmacological inhibitors or activators can be added to the neurons to understand their normal or pathological behavior. This method opens a new avenue for studying sympathetic biology and pathology since it is now possible to obtain biopsies of sympathetic neurons from humans easily.