Figure 3.

BMP4 Directs hESCs toward Proprioceptive-dI1 Sensory Interneurons

(A) BMP4 was added to neuralized EBs at day 6 (BMP4-D6), 8 (BMP4-D8), 10 (BMP4-D10), or 17 (BMP4-D17). EBs were collected at day 36 for IHC and RT-qPCR analyses to characterize the effects of BMP4 on dorsal neural identity.

(B and C) Transverse sections of E11.5 mouse lumbar (B) and day 28 monkey thoracic (C) embryonic spinal cord labeled with antibodies against β-III tubulin (red, Tuj1) and Lhx2 (green). The Lhx2⁺ Tuj1⁺proprioceptive dI1s originate in the dorsal-most spinal cord (B, box, B′).

(D–I) The addition of BMP4 at day 6 (E), 8 or 10 (F) resulted in elevated LHX2 expression (H) and significantly increased production of LHX2⁺ Tuj⁺ dI1s (E″, F″, and I) compared with RA controls (D″ and I; probability similar to RA control: p = 0.25, BMP4-D6; p < 0.043, BMP4-D8; p < 0.016, BMP4-D10); ∼20% of the BMP4-treated cells are LHX2⁺ (I). However, this effect is lost if BMP4 is added at day 17 (G, H, and I; probability similar to RA control, p = 0.20). There is no significance difference in LHX2 expression levels or numbers of LHX2⁺ Tuj⁺ dI1s among the BMP4-D6, BMP4-D8, and BMP4-D10 conditions (L, p = 0.77, BMP4-D6 versus BMP4-D8 versus BMP4-D10, one-way ANOVA). Note that in the RA condition, not all LHX2⁺ cells are Tuj1⁺ (arrows, D″). The region in the white dashed box in (D)–(G) is shown at higher magnification in (D′)–(G′) and (D″)–(G″).

Probability of similarity ^∗p < 0.05, ^∗∗∗p < 0.0005. Six biological replicates were performed, with 20–25 EBs quantified. The number of cells was normalized to the area of the EB and then scaled according to a unit area (10,000 μm²). Scale bar, 100 μm.