Three germ layer formation on micropatterns are extremely useful for quantitative analysis of hiPSC (human induced pluripotent stem cells) pluripotency. Spatial patterns of stem cells differentiated on the micropatterns will be formed from about 24 hours after differentiation induction and usually quantitated near 48 hours. To delineate the germ layer formation process, temporal changes in spatial patterning of germ layers should be analyzed by noninvasive microscopy. This study proposed a series of image processing methods combined with a U-net automatic segmentation to segment differentiated hiPSCs captured by bright-field microscopy. High segmentation accuracy (83.3%) for the test bright-field images compared with their concurrent Hoechst images (85%) was achieved. Tempo-spatial patterning and formation process of germ layers on the micropatterns can be visualized and quantified by segmenting time-lapse bright-field microscopy images using our method.