Generation and Characterization of Human Induced Pluripotent Stem Cell-derived Astrocytes Lacking Fragile X Messenger Ribonucleoprotein

This work focuses on four brain specific human astrocytes and how they're altered by neurodevelopmental disorders such as fragile X syndrome. Earlier work used animal models to study mostly neurons, so this work adds a new dimension to this area of research.

Mechanistic insights from animal studies have met with limited clinical success. The major challenge is to gain similar insights into neurons and astrocytes of human origin.

Our protocol successfully generated functional for wind specific astrocytes, which exhibit dysregulated, glycolytic, and mitochondrial metabolism in fragile X syndrome, enabling investigations into the role of glial cells in disease biology. Despite growing evidence on the key role of glial in neuronal function in health and disease, little is known about how human astrocytes are affected in neurodevelopmental disorders, such as fragile X syndrome.

Looking ahead, we'll build upon this framework to study how astrocytes and their interactions with neurons are altered by disease, and then how these may be corrected by therapeutic interventions.

[Instructor] To begin, coat a six-swell dish with Matrigel diluted at a ratio of one to 60 in advanced DMMF 12. Store the coated dish at a temperature of two to eight degrees Celsius. On the day of culturing, remove the coating material from the dish and add one milliliter of complete essential eight medium, along with a rock inhibitor at a final concentration of one X. Place the dish inside an incubator. Set at 37 degrees Celsius with 5% carbon dioxide before adding the human induced pluripotent stem cells, or IPSCs. Seed the human IPSCs in a complete essential eight medium with the rock inhibitor at a final concentration of one X to enhance colony adherence. The next day, replace the complete essential eight medium with fresh medium, lacking the rock inhibitor. Continue replenishing the medium daily until the cells reach 80% confluence, which takes approximately four to five days. When the colonies reach 80% confluence, add a mixture of collagenase and dispase to enzymatically detach them from the dish. Once the colonies begin to lift, remove the dish from the incubator and add two milliliters of DPBS to neutralize enzyme activity. Using DPBS, scrape off the colonies and collect the suspension into a 15 milliliter conical tube, using a wide board 10 milliliter serological pipette. Then, with a 10 milliliter serological pipette, gently triturate the suspension two to three times to break up the colonies. Allow the colonies to settle. After approximately two minutes, aspirate the DPBS enzyme mixture while leaving approximately one milliliter of liquid in the tube. Then add two milliliters of DPBS to the tube and mix by gently tapping to re-suspend the colonies. Allow them to settle, and repeat this process two times to remove residual enzyme. After removing the supernatant, re-suspend the colonies in one milliliter of complete essential eight medium and plate them into a freshly prepared Matrigel coated dish. Enzymatically lift the human induced pluripotent stem cells as described earlier. Plate them onto a non-adherent 100 millimeter suspension culture dish with chemically defined medium. Place the cell suspension on an orbital shaker at 40 revolutions per minute for seven days under normoxic conditions to facilitate the development of cortico spheres. On day eight, transfer the cortico spheres to a cell proliferation medium and maintain them for seven days. To induce glial specification, incubate the cortico spheres in glial enrichment medium for two weeks to obtain early glia spheres. For maturation of early glia spheres, replace basic fibroblast growth factor H with 20 nanograms per milliliter of leukemia inhibitory factor, and maintain the spheres for four weeks.

After four weeks in maturation medium, continue maintaining the spheres in glial enrichment medium for extended periods. To prevent aggregation and loss of viability, mechanically chop the glia spheres every two weeks, using a sterile industrial blade. Replace the entire medium with DNase one to remove DNA fragments generated from chopping. Next disassociate the glia spheres into mono layers of astrocyte progenitor cells, using a papain dissociation kit. Plate the dissociated cells onto a cell culture, treated adherent dish pre-coated with Matrigel at a one to 80 dilution. Propagate the astrocyte progenitor cells in glial enrichment medium until they reach 80% confluency. The passage the cells remove the spent medium and collect it in a conical tube. Add enzyme cell detachment medium to the cells and wait for one to two minutes. Once the cells begin to detach, add the spent medium to neutralize the enzyme activity. Collect the cell suspension and centrifuge at 800 G for two minutes. After discarding the supernatant, re-suspend the cells in glial enrichment medium. Plate approximately one times 10 to the power of six cells per well onto a Matrigel coated dish at a one to 80 dilution. For cryo-preservation, re-suspend the cell pellet in a cold mixture of 90% cortico sphere proliferation medium and 10% cryoprotectant. Transfer the re-suspended cells into cryo vials. Immediately move the cryo vials into a cryo box and store in a freezer at minus 80 degrees Celsius overnight. The following day, transfer all cryo vials to a liquid nitrogen tank for long-term storage. Characterize the dissociated astrocyte progenitor cells by immuno staining with Vimenton and nuclear factor IAM markers. To confirm the four brain regional specificity of human IPSC derived APCs, perform real-time QPCR analysis for human fork head box G1 and human homeo box, B4. Differentiate APCs into astrocytes and astrocytic differentiation medium for 14 days. Plate astrocytes on a 35 millimeter glass bottom dish at a density of five times 10 to the power of three cells per dish to measure their response to adenosine five tri phosphate. Allow the cells to adhere to the glass bottom for 24 hours. Wash the adhered cells three times with Hank's Balanced Salt Solution without calcium. Then incubate the cells in the culture medium containing five micromolar ratio metric diureide 2:00 AM, and 0.02% pluronic F127 for one hour at room temperature. After washing the cells twice with culture medium, replace the medium with two millimolar calcium containing Hank's Balanced Salt solution. Image the cells at two frames per second, using a 60X oil immersion objective on a focused drift compensating inverted microscope. Finally, record ATP induced calcium responses after adding ATP at a final concentration of five millimolar at the 25 second mark. Human IPSC colonies expressed pluripotent markers opt four and nano as confirmed by immuno staining. APCs derived from fragile X syndrome and control lines showed comparable proportions of vimentin and NFIA positive cells. Astrocyte progenitor cells from both control and fragile X syndrome human-induced pluripotent stem cells exhibited up-regulation of Fox G1, compared to HOXB4, indicating a forebrain identity. Astrocytes derived from human IPSC fragile X syndrome lines showed a reduced number of GFAP expression. Control and fragile X syndrome astrocytes exhibited ATP induced calcium transients, but the number of responders was significantly lower in fragile X syndrome astrocytes. The peak amplitude of the first ATP-evoked calcium transient was significantly lower in fragile X syndrome astrocytes compared to the control cells. Human IPSC derived fragile X syndrome astrocytes showed an increase in glycolysis, glycolytic capacity, and glycolytic reserves, but showed a significant decrease in maximal respiration in comparison to control astrocytes.