Self-Organization of Polarized Cerebellar Tissue in 3D Culture of Human Pluripotent Stem Cells

Abstract

During cerebellar development, the main portion of the cerebellar plate neuroepithelium gives birth to Purkinje cells and interneurons, whereas the rhombic lip, the germinal zone at its dorsal edge, generates granule cells and cerebellar nuclei neurons. However, it remains elusive how these components cooperate to form the intricate cerebellar structure. Here, we found that a polarized cerebellar structure self-organizes in 3D human embryonic stem cell (ESC) culture. The self-organized neuroepithelium differentiates into electrophysiologically functional Purkinje cells. The addition of fibroblast growth factor 19 (FGF19) promotes spontaneous generation of dorsoventrally polarized neural-tube-like structures at the level of the cerebellum. Furthermore, addition of SDF1 and FGF19 promotes the generation of a continuous cerebellar plate neuroepithelium with rhombic-lip-like structure at one end and a three-layer cytoarchitecture similar to the embryonic cerebellum. Thus, human-ESC-derived cerebellar progenitors exhibit substantial self-organizing potential for generating a polarized structure reminiscent of the early human cerebellum at the first trimester.

Is this a brain which I see before me? Modeling human neural development with pluripotent stem cells

“…For instance, mouse cortical progenitors are best differentiated from mouse ESCs in the presence of a chemical inhibitor of Shh signaling (Gaspard et al, 2008), whereas in otherwise similar culture conditions human PSCs convert efficiently to cortical precursors without Shh inhibition (Espuny-Camacho et al, 2013). Intriguingly, the species-specific requirement of Shh inhibition is similarly observed in the cerebellar models as well (Muguruma et al, 2015(Muguruma et al, , 2010Muguruma and Sasai, 2012;and see below). The origin of these differences remains unclear: whether they are related to differences in the levels of Shh signals or to differential sensitivity to Shh, or to other signals, will be interesting to explore further, as it could also be related to differences found in vivo, such as the relative sizes of the cerebral or cerebellar cortices.…”

“…Aside from their differential dependence on Shh signaling discussed above, the mouse and human models of cerebellar morphogenesis display additional differences (Muguruma et al, 2015(Muguruma et al, , 2010Muguruma and Sasai, 2012): the temporal progression of differentiation and functional maturation of cerebellar cell types takes two to three times longer with human cells and the final structure generated is much larger in size than with mouse cells (Fig. 2C).…”

“…A more complete and complex 3D model of early cerebellar development was recently described with mouse and human PSCs (Muguruma et al, 2015(Muguruma et al, , 2010 (Fig. 3B).…”

Translational potential of human brain organoids

“…Building on these earlier works, a recent flurry of papers described the generation of various neural organoids (Table 1), ranging from the whole‐brain organoids,27, 28, 29 to large sub‐brain regions such as cortical organoids,30, 31 to specific regions, including cerebellum,32 midbrain,33 adenohypophysis,34 hypothalamus,35 and hippocampus 36. Comparing with the classical neurospheres, these organoids are generally much larger.…”

3D culture models of tissues under tension

“…One classic example of such a system is the embryoid body that has been generated by culturing clusters of embryonic stem cells (or induced pluripotent stem cells) and allowed to differentiate into teratoma-like masses (Itskovitz-Eldor et al, 2000;Thomson et al, 1998). Although such systems have been used as models for generating early 'tissues', including developing brain structures (Muguruma et al, 2015(Muguruma et al, , 2010, maturation of tissues into their more adult forms and functions is also characterized by the substantial presence of extracellular matrix (ECM).…”