Fig. 3.

Directed spatial patterning in the microfluidic device reveals a region of permissive differentiation. Representative images and average plots of spatial differentiation of HB9⁺ cells (GFP labeled) along SHH gradient (n=4). Vertical bars on the left diagrammatically indicate the concentration and spatial gradient of RA and/or SHH. Plots to the right indicate the average intensity distribution from at least four experiments as well as actual PM concentrations based on computer simulations (quantified as mean percent cells/bin ±s.d., n=3). (A) Control HB9⁺ MNs subjected to a uniform concentration of PM and RA. Red dashed lines indicate example bin width. (B-D) HB9⁺ MNs subjected to varying PM gradients. Inset in C illustrates higher magnification detail of the MN cluster (200× confocal image). (E) The addition of an opposing gradient of BMP4 (20 ng/ml) further narrows the MN domain. (F) High expression of the pluripotency marker OCT4 towards the dorsal end of the microdevice (outside of the permissive MN region) indicates the effect of early exposure to a cross-gradient of BMP4. (G) Live/dead staining with Hoechst 33342 and propidium iodide (PI) reveal that we have not simply created a zone of permissive cell growth. *P≤0.05, **P≤0.01, ***P≤0.001.