Figure 1. Schematic for Human iPSC-Derived Midbrain Organoids

SCTi003-A iPSCs maintained in mTeSR™ Plus are dissociated and seeded at a density of 3 x 10^6 cells/well in seeding medium in AggreWell™800 plates. Thereafter, cultures are fed daily with formation medium. After 6 days, the organoids are transferred and cultured in suspension, allowing growth and subsequent patterning to the midbrain. Long-term maintenance and further maturation of midbrain organoids can be achieved using STEMdiff™ Neural Organoid Maintenance Kit, see the Product Information Sheet (PIS) for details. iPSC = induced pluripotent stem cell

Figure 2. Human iPSC-Derived Midbrain Organoids Exhibit High-Quality Homogeneous Morphology

Representative phase contrast images of midbrain organoids derived from SCTi003-A iPSCs using STEMdiff™ Midbrain Organoid Differentiation Kit show uniform size and spherical morphology at (A) Day 25, (B) Day 43, and (C) Day 120 in vitro. iPSC = induced pluripotent stem cell

Figure 3. Human iPSC-Derived Midbrain Organoids Express Key Markers of Brain-Region-Specific Patterning

Single organoids differentiated from SCTi003-A iPSCs were harvested for RNA at Day 60 and Day 100. Fold change is reported relative to parent hPSC and the TBP housekeeping gene control. (A) Midbrain floorplate precursor marker FOXA2 was elevated in midbrain organoids at Day 60 and Day 100. More mature marginal zone dopaminergic markers, (B) TH, (C) PITX3, and general neuronal marker (D) MAP2 were elevated at Day 60 and Day 100. (E) Glial marker GFAP expression is elevated in the organoids at Day 100. Colors represent separate differentiations. iPSC = induced pluripotent stem cell

Figure 4. Human iPSC-Derived Midbrain Organoids Exhibit Brain-Region-Specific Marker Expression and Organization

Representative images of (A) Day 60 and (B) Day 120 Human iPSC-Derived Midbrain Organoids. Midbrain organoids express general neuronal marker, MAP2, as well as elevated expression of floorplate progenitor marker, FOXA2, and more mature dopaminergic marker, TH.

Figure 5. Human iPSC-Derived Midbrain Organoids (Day 125) Demonstrate Treatment-Dependent Neuronal Activity Measured with the MEA Assay

Human iPSC-Derived Midbrain Organoids generated from SCTi003-A iPSCs using STEMdiff™ Midbrain Organoid Differentiation Kit were plated on an MEA plate (CytoView MEA 6, Axion Biosystems) and maintained with STEMdiff™ Neural Organoid Maintenance Medium. Activities from 64 electrodes were recorded from organoids at Day 125, after one hour of treatment with 4-AP, Rotenone, or DMSO as control, using a Maestro Pro MEA system™ (Axion Biosystems). (A) Representative brightfield image of a Human iPSC-Derived Midbrain Organoid on the MEA plate. (B-D) Detected spikes (black lines), single channel bursts (blue lines; a collection of at least 5 spikes, each separated by an ISI of no more than 100 ms), and network bursts (orange boxes; a collection of at least 50 spikes from a minimum of 35% of participating electrodes, each separated by an ISI of no more than 100 ms) were recorded for each treatment. Raster plots of spike activity show the Day 125 organoids exhibit increased network bursting upon 4-AP treatment (100 μM) and a decrease in network bursting with rotenone treatment (100 nM). Graphs comparing (E) mean firing rate, (F) network burst frequency, and (G) viability are shown. iPSC = induced pluripotent stem cell; MEA = multielectrode array; ISI = inter-spike interval