Generation of Induced Pluripotent Stem Cells from Turner Syndrome (45XO) Fetal Cells for Downstream Modelling of Neurological Deficits Associated with the Syndrome

Turner syndrome is a rare condition associated with a completely or partially missing X chromosome. The syndrome is characterized by infertility, short stature, cardiovascular disorders, and neurocognitive defects. In some cases, it causes fetal death.

Although somatic cells of aneuploid fetuses are biologically valuable, they are short lived, thus limiting their use in research. Generation of iPSC is thus an effective method of cell preparation for perpetual conservation of the aneuploid traits. They are self-renewing and can differentiate into specialized cell types reminiscent of early embryonic development.

Delivery of non-integrating episomal reprogramming plasmids through nuclear fiction is an efficient and reproducible method for generating completely reprogrammed and stable iPSC’s. This method can also be used on cells which are difficult to program. In this protocol we describe regeneration of iPSCs from fetal material with aneupliod.

Transfer the collected fetal chorionic villi to a sterile Petri dish. Wash it several times with PBS containing 1X antibiotic antimycotic solution. Remove the PBS completely by pipetting.

Add 1 ml collagenase blend to the villi and incubate at 37 degrees Celsius for 10 minutes or till the tissue disintegrates. After incubation, neutralize the collagenase by adding media containing 10%fetal Bovine Serum. Transfer the contents of the Petri dish into a 15 ml tube.

Centrifuge the digest to collect the disintegrated villi and released cells. Decant the supernatant carefully. Plate disintegrated villi along with released cells in a T25 culture flask and AmnioMAX media and grow till a confluent fibroblast culture is obtained.

Expand fibroblasts and culture to prepare stocks for use in subsequent transfection and characterization experiments. Prepare cultures of alkali containing the reprogramming plasmids. Isolate the plasmids using Midi plasmid purification kit, as per manufacturer’s instructions.

Re-suspend each pallet in suitable volume of DNAse RNAse free water to obtain a final concentration of 1 microgram per milliliter. Verify the plasmids using ECoR1 restriction digestion kit, following manufacturer’s instructions. Trypsinize the chorionic villi fibroblasts.

Neutralize, and centrifuge to collect the cells. Decant the supernatant, and re-suspend the cells in 5 ml OPTI-MEM. Count the cells using a hemocytometer, and remove 1 million cells for nucleofection.

Centrifuge to obtain cell pallet. Perform nucleofection using Amaxa NHDF Neucleofactor Kit. Prepare neucleofactor reagent by mixing 0.5 mL supplement and 2.25 mL nucleofactor solution provided in the kit.

Remove 100 microliters of nucleofactor solution, and add 1 microgram of each plasmid to it. Gently re-suspend 1 million cells in this mix. Transfer the cell DNA suspension into cuvette, ensuring that the sample covers the bottom of the cuvette without any air bubbles.

Cap the cuvette, and insert into the holder. Select the nucleofactor program D-23 for high efficiency, and apply. Remove cuvette out of the holder, and add 1 mL of AmnioMAX media.

Transfer the contents gently into a tissue culture treated Petri dish, using the pipette provided in the kit. Incubate the cells in a humidified carbon dioxide incubator at 37 degrees Celsius. Maintain the cells in AmnioMAX media for 10 days, and then shift to pluripotency medium for 20 days.

For iPSC expansion, manually dissect the embryonic stem cell-like colonies formed in the reprogramming dish using a pulled glass pipette. Propagate iPSCs appropriately, and massage every five to seven days. The iPCSs were positive for pluripotency marker alkaline phosphatase.

Generate embryoid bodies by cutting the iPSC colonies into small pieces and plating them in low attachment Petri dishes in embryoid body medium. Ectoderm differentiation is induced by plating day four embryoid bodies in ectoderm differentiation medium. Similarly, mesoderm differentiation is induced by plating day eight embryoid bodies in mesoderm differentiation medium.

For inducing endoderm differentiation, culture iPSCs in a monolayer in endoderm differentiation medium. Morphological changes in fibroblasts were monitored over the course of reprogramming. They express epithelial cell-like morphology and formed compact colonies.

The cells also had a height nucleus to cytoplasmic ratio, typical of pluripotent cells in culture. The iPSCs possessed a 45 XO carrier type and were positive for pluripotency markers OCT4, NANOG, SOX 2, SSEA 4, TRA-1-81 and E-CADHERIN. The cells were stained for representative germ-layer markers.

Turner Syndrome thus develops neurocognitive deficits. Neurons derived from Turner Syndrome iPSC can be an excellent model. This is modeled in a petri plate to understand the new line of normal disassociative Turner Syndrome.