Figure 1. Schematic for the STEMdiff™ Midbrain Organoid Differentiation Kit

hPSC-derived midbrain organoids can be generated in 43 days. Organoids are initially formed in AggreWell™800 plates. 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. hPSC = human pluripotent stem cell

Figure 2. Mature STEMdiff™ Midbrain Organoids Exhibit Brain-Region-Specific Marker Expression and Organization

Representative marker expression in Day 50 organoids produced using STEMdiff™ Midbrain Organoid Kits (left panels) is compared to organoids produced using STEMdiff™ Dorsal Forebrain Organoid Kits. (A) DAPI is used for counterstaining. (B) Midbrain organoids have comparatively less PAX6 expression throughout. Dorsal forebrain organoids exhibit elevated expression of dorsal cortical progenitor marker PAX6, organized radially. (C) Midbrain organoids have elevated expression of floorplate progenitor marker FOXA2 that arranges around pseudoventricular regions (see inset) and (D) marginal zone dopaminergic neuron marker GIRK2 that expands from FOXA2 expressing progenitor zones.

Figure 3. STEMdiff™ Midbrain Organoids Express Catecholaminergic Protein Tyrosine Hydroxylase

Midbrain organoids were generated from human ES cell line H7 and iPS cell line STiPS-F016 using STEMdiff™ Midbrain Organoid Differentiation Kit. Organoids were further matured to Day 50 with STEMdiff™ Neural Organoid Maintenance Kit and are compared to organoids generated with STEMdiff™ Dorsal Forebrain Organoid Differentiation Kit. Both guided organoid types generate neurons that express MAP2, but only midbrain organoids express the catecholaminergic neuron specific marker TH. ES cell = embryonic stem cell; iPS cell = induced pluripotent stem cell; TH = tyrosine hydroxylase

Figure 4. STEMdiff™ Midbrain Organoid Differentiation Kit Supports the Generation of Homogeneous, Reproducible Neural Organoids

(A-F) Representative phase contrast images of midbrain organoids derived from WLS-1C, H7, and STiPS-F016 hPS cell lines using STEMdiff™ Midbrain Organoid Differentiation Kit show uniform size and spherical morphology at Day 25 and Day 50 in vitro. (G) Diameter measurements of midbrain organoids demonstrate reproducibility across different cell lines (mean ± SD; Day 25, n = 21; Day 50, n = 26). hPS cell = human pluripotent stem cell

Figure 5. Neural Organoids Generated with STEMdiff™ Midbrain Organoid Kit Express Key Markers of Brain-Region-Specific Patterning

Single organoids differentiated from multiple cell lines were harvested for RNA at Day 25 and Day 50. ΔΔCT (Fold Change) of midbrain organoids is normalized to TBP and respective dorsal forebrain organoid control (A & B) or respective PSC control (C); mean ± SD, n = 3 - 15. (A) Midbrain floorplate precursor markers FOXA2, LMX1A, and EN1 were elevated in midbrain organoids at Day 25. (B) More mature marginal zone dopaminergic markers NURR1, TH, GIRK2, and PITX3 were elevated at Day 50. (C) Dorsal forebrain marker PAX6 and TBR1 expression is downregulated in Day 25 midbrain organoids. PSC = pluripotent stem cell