Fluidic Flow Enhances the Differentiation of Placental Trophoblast-Like 3D Tissue from hiPSCs in a Perfused Macrofluidic Device

The human placenta serves as a multifunctional organ to maintain the proper development of a fetus. However, our knowledge of the human placenta is limited due to the lack of appropriate experimental models. In this work, we created an in vitro placental trophoblast-like model via self-organization of human induced pluripotent stem cells (hiPSCs) in a perfused 3D culture macrofluidic device. This device allowed cell seeding, in situ trophoblast lineage differentiation, and formation of trophoblast-like tissues from hiPSCs in a biomimetic microenvironment. It incorporated extracellular matrix (ECM) and fluid flow in a single device. After trophoblast lineage differentiation, we were able to generate the 3D clusters with major cell types of the human placenta, including trophoblast progenitor cytotrophoblasts (CTBs), differentiated subtypes, syncytiotrophoblasts (STBs), and extravillous trophoblasts (EVTs) under long-term 3D culture (similar to 23 days). Moreover, the formed tissues exhibited enhanced expressions of CTB-, STB-, and EVT-related markers at the level of genes and proteins under a dynamic culture compared with static conditions. RNA-seq analysis revealed the higher expression of trophoblast-specific genes in 3D tissues, indicating the essential role of fluid flow to promote the trophoblast differentiation of hiPSCs. The established placental 3D model combined a bioengineering strategy with developmental principles, providing a promising platform for the study of placental biology in a biomimetic microenvironment in health and disease.