CAN EARLY FOREBRAIN PATTERNING/HOLOPROSENCEPHALY (HPE) BE MODELLED <EM >IN VITRO </EM>USING 2D AND/OR 3D HUMAN INDUCED PLURIPOTENT STEM CELL MODELS?

Holoprosencephaly (HPE) is the dysregulated midline cleavage of the developing forebrain. 14 HPE phenotypes exist, with ZIC2 (HPE5) and SIX3 (HPE2) being the most frequent mutations. This study examines whether forebrain patterning/HPE can be modelled using 2D and 3D hiPSC models. 2D monolayers were treated with different combinations of SMAD/WNT inhibitors, which resulted in a different pattern of neural PAX6 and SOX2 expression. 2D monolayers fed with neural induction media (NIM) only (no inhibitors) formed PAX6/SOX2 positive 3D spots. Characterisation of the 3D spots using immunofluorescence (IF) revealed increased ZIC2(HPE5) expression in the 3D spots compared to the 2D monolayer. In contrast, there was uniform expression of SIX3(HPE2) in both the 2D monolayer, and the 3D spots. Findings show GLI2 (HPE9) also had a higher expression in the 3D spots. Preliminary data suggests the 3D spots are anterior neural tissue (early forebrain spots) in a monolayer of anterior pre-placodal ectoderm. Following 5 days of NIM only or WNT antagonism, WNT signalling was activated or inhibited for 5 days in 2D monolayers. IF of day 10 monolayers confirmed the expression of FOXG1 and PAX6, suggesting forebrain identity. 3D spheroids generated using NIM with/without SMAD/WNT inhibitors, also displayed forebrain characteristic (FOXG1 positive IF). The 2D model expresses two of the most frequently mutated HPE genes, ZIC2 (HPE5) and SIX3 (HPE2), and can be used to study their regulation and function. Future directions include generating reporter hiPSC lines of SIX3 (HPE2) and ZIC2 (HPE3) to study their dynamic movement and understand spatial patterning.