Human induced pluripotent stem cells (iPSCs) are ideal for developing personalized medicine. However, the spontaneous differentiation of human iPSCs under conventional 2D and 3D cultures results in significant heterogeneity and compromised quality. Therefore, a method for effectively isolating and expanding high‐quality human iPSCs is critically needed. Here, a biomimetic microencapsulation approach for isolating and culturing high‐quality human iPSCs is reported. This is inspired by the natural proliferation and development of blastomeres into early blastocyst where the early embryonic stem cells‐containing core is enclosed in a semipermeable hydrogel shell known as the zona pellucida (Zona). Blastomere cluster‐like human iPSC clusters are encapsulated in a miniaturized (≈10 nanoliter) hyaluronic acid (HA)‐rich core of microcapsules with a semipermeable Zona‐like hydrogel shell and subsequently cultured to form pluripotent human iPSC spheroids with significantly improved quality. This is indicated by their high expression of pluripotency markers and highly efficient 3D cardiac differentiation. In particular, HA is found to be crucial for isolating the high‐quality human iPSCs with the biomimetic core‐shell microencapsulation culture. Interestingly, the isolated human iPSCs can maintain high pluripotency even after being cultured again in 2D. These discoveries and the bioinspired culture method may be valuable to facilitate the human iPSC‐based personalized medicine.
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