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. 2011 Nov 10;6(12):1920-8.
doi: 10.1038/nprot.2011.410.

Generating human intestinal tissue from pluripotent stem cells in vitro

Kyle W McCracken  1 , Jonathan C Howell, James M Wells, Jason R Spence
Affiliations

Generating human intestinal tissue from pluripotent stem cells in vitro

Kyle W McCracken et al. Nat Protoc. .

Abstract

Here we describe a protocol for generating 3D human intestinal tissues (called organoids) in vitro from human pluripotent stem cells (hPSCs). To generate intestinal organoids, pluripotent stem cells are first differentiated into FOXA2(+)SOX17(+) endoderm by treating the cells with activin A for 3 d. After endoderm induction, the pluripotent stem cells are patterned into CDX2(+) mid- and hindgut tissue using FGF4 and WNT3a. During this patterning step, 3D mid- or hindgut spheroids bud from the monolayer epithelium attached to the tissue culture dish. The 3D spheroids are further cultured in Matrigel along with prointestinal growth factors, and they proliferate and expand over 1-3 months to give rise to intestinal tissue, complete with intestinal mesenchyme and epithelium comprising all of the major intestinal cell types. To date, this is the only method for efficiently directing the differentiation of hPSCs into 3D human intestinal tissue in vitro.

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Conflict of interest statement

Competing Financial Interests Statement

J.M.W. and J.R.S. are inventors on a patent involving the system described herein.

Figures

Figure 1
Figure 1
Passaging hPSCs and differentiation to definitive endoderm. (a.) Schematic representation of the in vitro differentation of intestinal tissue from pluripotent stem cells. (b.) A low-power image of hPSCs (~85–90% confluent) in a 6-well dish that are ready to passage onto 24-well plates for differentiation. (c–e.) Higher-power images of hPSC colonies one day after plating at different split ratios (wells from 6-well dish: wells in 24-well dish). Cells were plated too dense (1:3) (c), at optimal density (1:6) (d) and too sparse (1:12) (e). (f–h.) Low-power image of hPSCs taken three days after plating. The cells plated at 1:6 dilution (g) are at an optimal density to begin differentiating to definitive endoderm. Note the spontaneous differentiation (arrows) in the well plated at 1:3 (f). (i–k.) Monolayers of definitive endoderm at the end of three-day Activin A treatment in different starting densities. Areas that appear to remain undifferentiated (dashed circle) are evident in the cultures that were plated at 1:3 (i). Scale bar in b and f–h corresponds to 2mm and in c–e and i–k to 200μm.
Figure 2
Figure 2
Spheroid formation from hPSC-derived definitive endoderm. Treatment of endoderm with Wnt3a+FGF4 induces hindgut differentiation and spheroid formation. Low-power images show cultures after 24 (a–c), 48 (d–f), 72 (g–i) and 96 hours (j–l) of treatment at different densities, too dense (1:3), optimal density (1:6) and too sparse (1:12). Scale bars correspond to 2mm.
Figure 3
Figure 3
Three-dimensional growth of intestinal organoids in matrigel. (a–f) Low-power growth series of organoids in matrigel bead. Inset in a shows high-power image of d0 spheroids (arrows). (g–j) High power images of two- to four-week old organoids. (i) A 28 day old organoid that is ready to be split. White dashed line denotes the plane along which the organoid will be cut. (j) A 28 day old organoid that was cut and cultured for an additional 7 days. White dashed line denotes the plane where the organoid was cut, and the arrowheads show new epithelial growth. Scale bar in a–f corresponds to 2mm and in a (inset) and g–j to 0.5mm.
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