Self-organized anteroposterior regionalization of early midbrain and hindbrain using micropatterned human embryonic stem cells

Abstract

To develop into the central nervous system, neuroepithelial cells must first form a neural tube consisting of a series of patterned neural progenitor cells along the anterior-posterior (AP) axis. Based on studies using model organisms, it has been revealed that AP spatial regionalization is dominated by gradients of morphogens that regulate retinoic acid (RA), sonic hedgehog (SHH), bone morphogenetic proteins (BMPs), and Wingless/int1 (WNT) signaling pathways. Recently, human pluripotent stem cells (hPSCs) were successfully induced into a patterned neural tissue with differential AP gene expression levels by a gradient of WNT activity controlled by a microfluidic device. However, the midbrain and hindbrain boundaries were not as sharp as observed in vivo, likely due to the lack of additional important morphogenic factors, such as RA and SHH. Here, we induced micropatterned hPSCs into AP patterned neural tissue by activating not only WNT but also RA and SHH signals under fully defined culture conditions. We found that hPSCs self-organized into spatially patterned midbrain (FOXG1-OTX2+) and hindbrain (HOXB4+) progenitors with a sharp boundary after 6 days of induction. Following the initial induction, the cells with midbrain identities near the pattern boundary folded inwardly to form a 3D structure, maintaining a distinct boundary between OTX2+ and HOXB4+ zones. To investigate the mechanism of cell fates patterning, we found that the reaction-diffusion of BMP/Noggin played a role in AP regionalization, while differential mechanical stress and cell sorting were unlikely to be involved. Then, we validated our model by investigating the effects of exposure to two known teratogens including valproic acid and isotretinoin. Drug treatment results successfully predicted that valproic acid inhibited the development of both midbrain and hindbrain development while isotretinoin disrupts the normal AP patterning of the midbrain and hindbrain. In conclusion, by integrating engineering approaches and chemically defined culture conditions, we have developed an in vitro AP patterned model of early human midbrain and hindbrain development, and we have revealed its potential to be employed as a high throughput drug discovery system.