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Background: The differentiation of human pluripotent stem cells (hPSC) into intestinal organoids represent an attractive mechanism for generating cells for disease modelling, drug screening and cell-replacement therapy. Although it is possible to generate intestinal organoids from adult stem cells, the derivation of organoids from hPSCs has been shown to result in a broader variety of cells as the starting material. Aims: Introduce novel PSC to intestinal differentiation kit and protocols. Methods: We developed the STEMdiffTM Intestinal Organoid Kit, a specialized serum-free and fully-defined medium formulation that efficiently and reproducibly promotes differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells through developmental stages of 1) definitive endoderm, 2) mid-/hindgut, and 3) small intestine. Results: Here we demonstrate that monolayers generated with multiple human ES (WA01, WA07, WA09) and iPS (WLS-1C, STiPS-FO16, STiPS-MOO1) lines maintained in mTeSRTM with Corning MatrigelTM, differentiate into FOXA2 + /SOX17 + endoderm cultures with an efficiency of 81.6% ± 8.6% (n=21). Further differentiation into posterior endoderm to promote the formation of mid-/hindgut resulted in 71.4 ± 8.5% (n=13) of cells expressing the hindgut marker CDX2, but none of the cells expressing the anterior gut tube marker SOX2. Twenty-four hours after the emergence of CDX2 + cells, clusters in the flat cell sheet monolayers changed their morphology to tightly packed epithelial tubes that generated budding spheroids which detached from the monolayer. These detached hindgut spheroids are composed of CDX2 + /E-cadherin +

A164 HIGHLY EFFICIENT DIFFERENTIATION OF HUMAN PLURIPOTENT STEM CELLS INTO LONG-TERM EXPANDABLE “MINI-GUT” ORGANOIDS