Int J Stem Cells.  2022 Feb;15(1):104-111. 10.15283/ijsc21209.

Generation of Highly Expandable Intestinal Spheroids Composed of Stem Cells

• Kwon O
• Kwon O
• Yu WD
• Yu WD
• Son YS
• Son YS
• Jung KB
• Jung KB
• Lee H
• Lee H
• Son MY
• Son MY

• KMID: 2526742
• DOI: http://doi.org/10.15283/ijsc21209

Abstract

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.

Keyword

Human intestinal organoid (HIO); Expandable Intestinal spheroid (InS^exp ); Intestinal stem cell; WNT pathway; Maturity memory

Figure

• 

  Fig. 1 Culture and characterization of InSexp derived from 3D HIOs. (A) Schematic diagram of the InSexp culture method. (B, C) Bright field images of InSexp by data, after thawing (B), and by passaging (C). White scale bars, 500 μm. Yellow scale bars, 250 μm. (D) Relative expression of intestinal epithelial cell marker genes in hPSCs, InSexp, and HIOs. (E) Immunofluorescence analysis of intestinal epithelial cell markers in InSexp. White scale bars, 50 μm. (F) Bright field images of 3D HIOs, InSexp, and re-embedded InSexp. White scale bars, 500 μm. Yellow scale bars, 250 μm. *p<0.05, **p<0.01, ***p<0.001 using two-tailed t-test.

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  Fig. 2 CHIR99021 treatment promotes growth rate of InSexp. (A, B) Effect of CHIR99021 on the InSexp growth rate at multiple concentrations. Bright field images of the InSexp (A) and Relative fold change in the InSexp cell number (B). White scale bars, 500 μm. Data represent means±SEM (n=4). (C, D) Recovery of InSexp-forming competency by addition of CHIR99021 in WNT3A- and R-spondin 1-depleted medium. Bright field images of InSexp (C) and Relative fold change in the InSexp cell number (D). White scale bars, 500 μm. Yellow scale bars, 250 μm. Data represent means±SEM (n=4). (E) Inhibition of InSexp growth by treatment with WNT inhibitors. White scale bars, 500 μm. Data represent means±SEM (n=4). (F) Immunofluorescence analysis and quantification of proliferating cell markers in InSexp. Scale bars, 200 μm. Data represent means±SEM (n=5). *p<0.05, **p<0.01, ***p<0.001 using two-tailed t-test.

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  Fig. 3 Characterization of InSexp derived from immature and mature 3D HIOs. (A, B) Effect of CHIR99021 on InSexp derived from immature and mature HIOs. Bright field images of InSexp (A) and cell number increase of the InSexp after day 14 (B). White scale bars, 500 μm. Yellow scale bars, 250 μm. Data represent means±SEM (n=4). (C) Relative gene expression of intestinal epithelial cell markers and WNT target genes in InSexp derived from immature and mature HIOs. (D, E) Immunofluorescence analysis of the intestinal epithelial cell markers and WNT target genes in InSexp derived from immature and mature HIOs. White scale bars, 50 μm. *p<0.05, **p<0.01, ***p<0.001 using two-tailed t-test.

Reference

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