Journal: SLAS technology

Article Title: Self-organized anteroposterior regionalization of early midbrain and hindbrain/spinal cords using micropatterned human pluripotent stem cells

doi: 10.1016/j.slast.2026.100419

Figure Lengend Snippet: ( A ) Representative phase-contrast and immunostaining fluorescence images showing the spatially patterned expression of anterior (OTX2) and posterior (HOXB4) neural markers on day 6 and day 7. The OTX2 and HOXB4 merged image was shown. Cell nuclei were counterstained with DAPI. ( B ) Colorimetric maps showing the average fluorescent intensity of OTX2 and HOXB4 staining. The intensity of each pixel of the images was normalized to the maximum value for each image. n > 15. ( C ) Plots showing the quantitative average intensity in relation to the distance to the center of the pattern. ( D ) Representative confocal images and the reconstructed side view showing the three-dimensional architecture and the spatial expression of OTX2+ and HOXB4+ cells on day 7. Cell nuclei were counterstained with DAPI. ( E ) A schematic showing the self-organized anteroposterior patterning generated from human ESCs as well as the folding process and 3D structural change from day 6 to day 7. In these experiments, H9 human ESCs were differentiated with the induction protocol. Scale bar, 100 μm.

Article Snippet: The primary antibodies used in this study include HoxB4 (rat, DSHB, I12, 1:20), OTX2 (goat, R&D Systems, 1:100), and PAX6 antibodies (mouse, Abcam, Ab78545, 1:100).

Techniques: Immunostaining, Fluorescence, Expressing, Staining, Generated

Journal: SLAS technology

Article Title: Self-organized anteroposterior regionalization of early midbrain and hindbrain/spinal cords using micropatterned human pluripotent stem cells

doi: 10.1016/j.slast.2026.100419

Figure Lengend Snippet: ( A ) Representative immunostaining fluorescence images showing the spatial expression of neural markers: OTX2 and HOXB4 at 136 h and 140 h. The OTX2 and HOXB4 merged image was shown. ( B ) Representative immunostaining fluorescence images showing the spatial expression of OTX2 and HOXB4 for the DMSO control group, Blebbistatin (a myosin inhibitor, 10 μM) treated group, and Y27632 (a ROCK inhibitor, 10 μM) treated group. The OTX2 and HOXB4 merged image was shown. ( C ) Colorimetric maps showing the average fluorescent intensity of OTX2 and HOXB4 staining. The intensity of each pixel of the images was normalized to the maximum value for each image. n > 15. ( D ) Plots showing the quantitative average intensity of OTX2 and HOXB4 in relation to the distance to the center of the pattern. Scale bar, 100 μm.

Article Snippet: The primary antibodies used in this study include HoxB4 (rat, DSHB, I12, 1:20), OTX2 (goat, R&D Systems, 1:100), and PAX6 antibodies (mouse, Abcam, Ab78545, 1:100).

Techniques: Immunostaining, Fluorescence, Expressing, Control, Staining

Journal: SLAS technology

Article Title: Self-organized anteroposterior regionalization of early midbrain and hindbrain/spinal cords using micropatterned human pluripotent stem cells

doi: 10.1016/j.slast.2026.100419

Figure Lengend Snippet: ( A ) Representative immunostaining fluorescence images showing the spatial expression of OTX2 and HOXB4 across micropatterns on day 6 with diameters of 200 μm, 400 μm, 800 μm, or 1000 μm. ( B ) Representative immunostaining fluorescence images showing the spatial expression of OTX2 and HOXB4 of human ESCs induced with a lower concentration (1 ng/ml) or a higher concentration (10 ng/ml) of BMP4. ( C ) Colorimetric maps showing the average fluorescent intensity of OTX2 and HOXB4 staining. The intensity of each pixel of the images was normalized to the maximum value for each image. n > 15. ( D ) Plots showing the quantitative average intensity of OTX2 and HOXB4 in relation to the distance to the center of the pattern. Scale bar, 100 μm.

Article Snippet: The primary antibodies used in this study include HoxB4 (rat, DSHB, I12, 1:20), OTX2 (goat, R&D Systems, 1:100), and PAX6 antibodies (mouse, Abcam, Ab78545, 1:100).

Techniques: Immunostaining, Fluorescence, Expressing, Concentration Assay, Staining

Journal: SLAS technology

Article Title: Self-organized anteroposterior regionalization of early midbrain and hindbrain/spinal cords using micropatterned human pluripotent stem cells

doi: 10.1016/j.slast.2026.100419

Figure Lengend Snippet: ( A ). Representative immunostaining fluorescence images showing the spatial expression of OTX2 and HOXB4 of human ESCs induced with a lower concentration (0.1 μM) or a higher concentration (1.5 μM) of all-trans retinoic acid (RA) on day 6. ( B ). Colorimetric maps showing the average fluorescent intensity of OTX2 and HOXB4 staining. The intensity of each pixel of the images was normalized to the maximum value for each image. n > 15. ( C ). Plots showing the quantitative average intensity of OTX2 and HOXB4 in relation to the distance to the center of the pattern. ( D ). Representative immunostaining fluorescence images showing the spatial expression of OTX2 and HOXB4 of human ESCs induced with a lower concentration (30 nM) or a higher concentration (600 nM) of WNT activator CHIR99021 on day 6. ( E ). Colorimetric maps showing the average fluorescent intensity of OTX2 and HOXB4 staining. The intensity of each pixel of the images was normalized to the maximum value for each image. n > 15. ( F ). Plots showing the quantitative average intensity of OTX2 and HOXB4 in relation to the distance to the center of the pattern. Scale bar, 100 μm.

Article Snippet: The primary antibodies used in this study include HoxB4 (rat, DSHB, I12, 1:20), OTX2 (goat, R&D Systems, 1:100), and PAX6 antibodies (mouse, Abcam, Ab78545, 1:100).

Techniques: Immunostaining, Fluorescence, Expressing, Concentration Assay, Staining

Journal: SLAS technology

Article Title: Self-organized anteroposterior regionalization of early midbrain and hindbrain/spinal cords using micropatterned human pluripotent stem cells

doi: 10.1016/j.slast.2026.100419

Figure Lengend Snippet: ( A ) Representative immunostaining fluorescence images showing the spatial expression of OTX2 and HOXB4 in induced human ESCs treated with DMSO (control) and medium dosages of penicillin G, valproic acid, and isotretinoin on day 6. ( B ) Colorimetric maps showing the average fluorescent intensity of OTX2 and HOXB4 staining. The intensity of each pixel of the images was normalized to the maximum value for each image. n > 15. ( C ) Plots showing the quantitative average intensity in relation to the distance to the center of the pattern. ( D ) Representative immunostaining fluorescence images showing the spatial expression of OTX2 and HOXB4 in induced human ESCs treated with DMSO (control) and medium dosages of penicillin G, valproic acid, and isotretinoin on day 7. ( E ) Colorimetric maps showing the average fluorescent intensity of OTX2 and HOXB4 staining. The intensity of each pixel of the images was normalized to the maximum value for each image. n > 15. ( F ) Plots showing the quantitative average intensity in relation to the distance to the center of the pattern. ( G ) The percentage of area covered by OTX2+ midbrain in DMSO control, penicillin G, VPA, and isotretinoin treated groups on day 6. Data are represented as mean ± s .e.m. n.s. (no significance), P > 0.05;***, P < 0.001. Scale bar, 100 μm.

Article Snippet: The primary antibodies used in this study include HoxB4 (rat, DSHB, I12, 1:20), OTX2 (goat, R&D Systems, 1:100), and PAX6 antibodies (mouse, Abcam, Ab78545, 1:100).

Techniques: Immunostaining, Fluorescence, Expressing, Control, Staining

Journal: SLAS technology

Article Title: Self-organized anteroposterior regionalization of early midbrain and hindbrain/spinal cords using micropatterned human pluripotent stem cells

doi: 10.1016/j.slast.2026.100419

Figure Lengend Snippet: The model features circularly micropatterned human pluripotent stem cells (hPSCs) that, by day 6, self-organize into distinct zones. These zones exhibit anterior-posterior (AP) patterning of FOXG1-FOXA1+OTX2+ ventral midbrain and HOXB4+ hindbrain/spinal cord fates. The tissue then undergoes inward folding to form a three-dimensional (3D) annular structure while maintaining a clear boundary between the OTX2+ and HOXB4+ zones. The reaction-diffusion of BMP/Noggin plays a key role in the mechanism of midbrain and hindbrain/spinal cord fate patterning. This model can be potentially used in drug testing, which is demonstrated by its capability to differentiate the teratogenic effects of drugs like valproic acid (Depakene) and isotretinoin. VPA treatment impairs the development of both midbrain and hindbrain/spinal cord fates, delays anterior-posterior patterning formation on day 6, and leads to 3D folding failure on day 7. Interestingly, isotretinoin treatment leads to a random mix of OTX2+ and HOXB4+ cells, obliterating the anterior-posterior patterning and the formation of 3D structures. Compared to 2D cell culture, the unique phenotypes including completely separated midbrain/hindbrain/spinal cord zones and tissue folding can be used to quantify developmental defects. This innovative model offers an excellent experimental platform for exploring human brain development mechanisms and enhancing drug testing methodologies.

Article Snippet: The primary antibodies used in this study include HoxB4 (rat, DSHB, I12, 1:20), OTX2 (goat, R&D Systems, 1:100), and PAX6 antibodies (mouse, Abcam, Ab78545, 1:100).

Techniques: Diffusion-based Assay, Cell Culture