Patient-Derived Midbrain Organoids to Explore the Molecular Basis of Parkinson's Disease

Abstract

Induced pluripotent stem cell-derived organoids offer an unprecedented access to complex human tissues that recapitulate features of architecture, composition and function of in vivo organs. In the context of Parkinson's Disease (PD), human midbrain organoids (hMO) are of significant interest, as they generate dopaminergic neurons expressing markers of Substantia Nigra identity, which are the most vulnerable to degeneration. Combined with genome editing approaches, hMO may thus constitute a valuable tool to dissect the genetic makeup of PD by revealing the effects of risk variants on pathological mechanisms in a representative cellular environment. Furthermore, the flexibility of organoid co-culture approaches may also enable the study of neuroinflammatory and neurovascular processes, as well as interactions with other brain regions that are also affected over the course of the disease. We here review existing protocols to generate hMO, how they have been used so far to model PD, address challenges inherent to organoid cultures, and discuss applicable strategies to dissect the molecular pathophysiology of the disease. Taken together, the research suggests that this technology represents a promising alternative to 2D in vitro models, which could significantly improve our understanding of PD and help accelerate therapeutic developments.

Keywords: IPS (induced pluripotent stem) cell; Parkinson's disease; dopamine; genetics; midbrain; organoid.

Figure 1

Summarized hMO differentiation strategies. hMO differentiation protocols aim at reproducing essential dynamics of in vivo human midbrain development, which are represented by the drawings in the middle section: (1) midbrain specification using dual SMAD inhibition and WNT modulation; (2) midbrain floor plate (mfp) induction through modulation of SHH and FGF8, and (3) differentiation and maturation of midbrain dopaminergic neurons using neurotrophic factors (see Table 1 for details in hMO models of PD). Each step is associated with the generation of cell types that can be identified in vivo and in hMO models using the mentioned markers. Starting from hiPSC-derived embryonic bodies (EB), the described protocols have either relied on the use of sequential or simultaneous patterning strategies, represented as the top and bottom branches, respectively. Optional modifications to the protocol include expansion of mfpNeural Progenitor Cells (mfpNPC), hydrogel embedding, use of orbital shakers or microfluidics devices, aging strategies, and DA treatment. The generated hMO typically contain mDA neurons expressing markers of A9 and A10 terminal differentiation, and long-term cultures may favor the apparition of neuromelanin granules, which can be enhanced through DA stimulation. Features of floor plate organization in ventricle, intermediate, and mantle zones (VS, IZ, MZ) may also be revealed using markers of mDA progenitors (65). This organization is particularly evident in hydrogel-embedded organoids, as this process favors apico-basal polarization. SB, SB431542; DM, dorsomorphin; CHIR, CHIR99021; A83, A-83-01; SHH, sonic hedgehog; SAG, smoothened agonist; FGF8: fibroblast derived growth factor 8; BDNF/GDNF, brain/glial-derived neurotrophic factor; AA, ascorbic acid; cAMP, cyclic AMP; LDN, LDN193189; PM, purmorphamine; TGFß3, Transforming growth factor beta 3; FGF20, fibroblast derived growth factor 20.

Figure 2

Developments and applications of hMO cultures. (A) Potential developments of hMO cultures include aging strategies, fusions with other brain region organoids, co-cultures with non-neuronal lineage cells (such as microglia, endothelial cells), use of microfluidics or “organ on a chip” approaches, and in vivo transplantations. (B) hMO constitute relevant biological platforms to study the effects of PD-associated genetic and environmental factors on cellular function and molecular networks. Such approaches may lead to a better understanding of the molecular basis of PD, help identify new therapeutic targets, and develop personalized medicine approaches.