3D Cerebellar Differentiation and Purkinje Cell Maturation from Human Induced Pluripotent Stem Cell

The cerebellum plays a critical role in maintenance of balance and posture, coordination of voluntary movements and motor learning. Cerebellar ataxias represent a heterogeneous group of disorders characterized by progressive neurodegeneration of the cerebellum for which no cure is currently available. Most of the current knowledge about ataxias is based on postmortem studies and animal models that do not fully recapitulate the features of human disease. The development of new models to study cerebellar ataxia is therefore an important unmet medical need. In recent years, discoveries regarding the mechanism of cerebellar differentiation has promoted the generation of cerebellar neurons from pluripotent stem cells¹. However, despite recent progress, the establishment of long-term culture systems and generation of different mature cerebellar neurons remains a challenge.

In the present study, we recapitulate the early developmental events of human cerebellum in vitro from human iPSCs in 3D, with generation of continuous cerebellar plate neuroepithelium. For this propose, cells were differentiated into cerebellar neurons using a 3D culture system and sequential addition of growth factors, including FGF2, FGF19 and SDF1¹. FGF2 has an inductive role in cerebellar differentiation, with a major effect in the caudalization of neuroepithelium tissue. FGF19 was necessary for promoting spontaneous generation of rostral hindbrain neural-tube-like structures with dorsal-ventral (D-V) polarity, and SDF1 was shown to facilitate spontaneous generation of rhombic lip-like structure as seen at the developmental stage when cerebellar neurogenesis occurs. Our system generated a neuroepithelium organized into a multilayered structure with basal-apical polarity, including Rhombic-lip-derivative zone (BARHL1+), Purkinje cells precursors zone (Olig2+ and SKOR2+) and Ventricular zone (Sox2+ and N-cad+), characteristic to the cerebellar ontogenesis. Furthermore, we are able to maintain this 3D culture until 134 days of differentiation and obtain MAP2⁺ and calbindin⁺ mature neurons.

Given the importance of Purkinje cells in cerebellar neuropathology, we proceeded to isolate these cells without the need for co-culture with other cell types or cerebellum slices, as has been previously demonstrated^1,2. To this end we dissociated 3D organoids and replated the cells on laminin in a defined serum-free neuronal basal medium reported to be more representative of the central nervous system extracellular environment and increasing cell survival and the proportion of synaptically active neurons³. Replated cells show typical markers of Purkinje cells (calbindin), and were able to organize into intricate neuronal networks. Cells were maintained for 134 days in culture and show signs of maturation.

To our best knowledge, this is the first report that shows the culture of Purkinje cells without co-culture and using defined conditions. Thus, we have established an important foundation to generate cultures of cerebellar neurons that can be used for further studies on cerebellar ataxias.

1. Muguruma, K., Nishiyama, A., Kawakami, H., Hashimoto, K. & Sasai, Y. Self-Organization of Polarized Cerebellar Tissue in 3D Culture of Human Pluripotent Stem Cells. Cell Rep. 10, 537–550 (2015).

2. Wang, S. et al. Differentiation of human induced pluripotent stem cells to mature functional Purkinje neurons. Sci. Rep. 5, 9232 (2015).

3. Bardy, C. et al. Neuronal medium that supports basic synaptic functions and activity of human neurons in vitro. Proc. Natl. Acad. Sci. U. S. A. 112, E2725–34 (2015).