Generation of Highly Expandable Intestinal Spheroids Composed of Stem Cells

Many of early findings regarding intestinal stem cells (ISCs) and their niche in the human intestine have relied on colorectal cancer cell lines and labor-intensive and time-consuming mouse models. However, these models cannot accurately recapitulate the physiologically relevant aspects of human ISCs. In this study, we demonstrate a reliable and robust culture method for 3D expanding intestinal spheroids (InS(exp)) mainly comprising ISCs and progenitors, which can be derived from 3D human intestinal organoids (HIOs). We did functional chararcterization of InS(exp) derived from 3D HIOs, differentiated from human pluripotent stem cells, and optimization culture methods. Our results indicate that InS(exp) can be rapidly expanded and easily passaged, and show enhanced growth rates via WNT pathway activation. InS(exp) are capable of exponential cell expansion and cryopreservation. Furthermore, in vitro-matured HIO-derived InS(exp) proliferate faster than immature HIO-derived InS(exp) with preservation of the parental HIO characteristics. These findings may facilitate the development of scalable culture systems for the long-term maintenance of human ISCs and provide an alternative platform for studying ISC biology.