The H9 human embryonic stem cells (hESCs) were purchased from WiCell Research Institute (Madison, WI, USA). Human induced pluripotent stem cells (hiPSCs) were generated as described previously (6).

hPSC-derived 3D HIOs were generated as reported previously (1, 2). The 3D HIOs were mechanically dissociated and re-embedded every 10∼14 days. For in vitro maturation of HIOs, 1 ng/ml of IL-2 (R&D systems) was added to the culture medium when the spheroids were embedded. IL-2 was freshly added after at least two passages.

The 3D InS^exp derived from HIOs were generated as previously reported (7). The growth medium was replaced every other day. To optimize the organoid growth, 1 μM of CHIR99021 (Tocris) was included in the InS^exp growth medium.

For passaging, InS^exp were dissociated using trypsin-EDTA and a P1000 pipette was used to break up the Matrigel dome. The total mixture was transferred to a conical tube, incubated at 37℃ in a water bath for 5 min, and mechanically dissociated using 10 rounds of pipetting. The dissociated cells were resuspended and centrifuged at 1,500 rpm, 4℃, for 5 min. After resuspending the cell pellet with Matrigel, the HIO-Matrigel mixture was plated onto a four-well culture plate and incubated at 37℃ in a CO₂ incubator for more than 30 min to solidify the Matrigel, followed by the addition of InS^exp growth medium supplemented with 10 μl of Y-27632 (Tocris), and 1 μM Jagged-1 (AnaSpec, Fremont, CA, USA) for the first 2 days. Subsequently, this medium was replaced with the InS^exp culture medium, which was then replaced every other day.

Total RNA was extracted from cells using the RNeasy kit (Qiagen), and cDNA was prepared using a Superscript IV cDNA synthesis kit (Thermo Fisher Scientific Inc.). Quantitative polymerase chain reaction (qPCR) was performed using a 7500 Fast Real-Time PCR system (Applied Biosystems, Foster city, CA, USA). Relative expression was calculated using the ΔΔCt method. The primers used in this study are listed in Table S1.

Immunofluorescence staining was performed on 10% formalin-fixed InS^exp as described previously (8). Cryosections were permeated with 0.1% Triton X-100, and then blocked for 1 hour with 4% bovine serum albumin. After blocking, the sections were incubated with primary antibodies at 4℃ overnight and the secondary antibody at room temperature. Finally, 4’,6-diamidino-2-phenylindole (DAPI; 1 mg/ml, Thermo Fisher Scientific Inc.) was used to counterstain the nuclei. Fluorescence images were acquired using a fluorescence microscope (IX51, Olympus, Tokyo, Japan) and LSM800 conforcal microscope (Carl Zeiss, Gottingen, Germany). The list of primary antibodies used is provided in Table S2.

The InS^exp were dissociated to single cells using 0.25% trypsin-EDTA for 10 min at 37℃. The dissociated cells were then diluted with advanced DMEM/F12 basal media and centrifuged for 5 min at 1,500 rpm. The single cells were resuspended in InS^exp growth medium and stained with trypan blue. The cell counts and sizes were determined using the Countess 3Automated Cell Counter (Thermo Fisher Scientific Inc.).

All experiments were repeated at least three times and results are presented as means±standard error (SEM). Two-tailed student’s t-test was performed to determine the significance of the data statistically.

To develop a fast expandable 3D ISC culture method, we dissociated hPSC-derived HIOs into small fragments and embedded them in a Matrigel dome (Fig. 1A). Embedded cells formed morphologically distinct structures compared to HIOs and showed a hollow spherical shape (Fig. 1B). This new type of 3D intestinal organoids, designated as InS^exp, is highly proliferative and easily passaged by single cell dissociation for more than 6 months without spontaneous differentiation and cell death (Fig. 1C). The InS^exp is mostly composed of ISCs and progenitors, and thus exhibits higher expression of stem cell/progenitor marker genes and proteins (Fig. 1D and 1E). Furthermore, the InS^exp can be cryopreserved without any loss of morphology and function upon thawing (Fig. 1B). These data suggest that the newly developed InS^exp culture method enables rapid expansion and cryopreservation through the enrichment of ISCs and progenitors. Interestingly, ISC and progenitor-enriched InS^exp further differentiated into 3D HIOs by re-embedding in a new Matrigel without cell dissociation, and the generation of multiple budding structures in the re-embedded InS^exp is similar to the structural characteristics of the original 3D HIOs (Fig. 1F).

As treatment with CHIR99021 has been reported to enhance the growth of mouse and human adult stem cell-derived intestinal organoids (9, 10), we supplemented a glycogen synthase kinase 3 (GSK-3) inhibitor CHIR99021 in the InS^exp culture medium to optimize the culture medium. First, we performed a titration experiment with increasing concentrations of CHIR99021 to assess the optimal concentration range of WNT activator. The growth of InS^exp was maximized by treatment with 1 μM CHIR99021; however, InS^exp growth was suppressed when treated with CHIR99021 at a concentration higher than 1 μM (Fig. 2A and 2B). Interestingly, treatment with CHIR99021 at more than 6 μM completely inhibited the growth of InS^exp (Fig. 2A and B). As the growth of InS^exp was maximized when treated with 1 μM of CHIR99021, this was the concentration used in all subsequent experiments. We next examined the effect of WNT ligand depletion on the growth of InS^exp. As expected, the growth rate of InS^exp was significantly decreased by depletion of WNT ligands, WNT3A and R-spondin 1, from the growth medium, and was mostly recovered by supplementation with CHIR99021 (Fig. 2C and 2D). In line with this, the spheroid formation and growth of InS^exp were completely diminished by treatment with WNT pathway inhibitors such as WNT C-59 and XAV939 (Fig. 2E). Therefore, these data indicate that the WNT pathway plays an essential role in controlling the growth of InS^exp, and that the growth of InS^exp can be enhanced by moderate activation of the WNT pathway using 1 μM of CHIR99021. The WNT pathway was found to be essential for InS^exp proliferation, as KI67^＋ proliferating cells disappeared under WNT-depleted or WNT inhibitor-treated conditions; however, these did not induce cell death (Fig. 2F). Taken together, we suggest that moderate activation of the WNT pathway augments the growth rate of InS^exp by increasing cell proliferation, and that the growth medium was optimized by adding a low concentration of CHIR99021.

We previously demonstrated that hPSC-derived hIOs can be matured in vitro using interleukin-2 treatment, and these were more similar to the adult human small intestine (2). Therefore, we investigated whether 3D InS^exp derived from immature or in vitro-matured HIOs exhibited distinct marker gene expression pattern and functional characteristics. First, when the same number of cells was embedded in a Matrigel dome, the spheroid formation efficiency of mature HIO-derived InS^exp was significantly higher than that of immature HIO-derived InS^exp (Fig. 3A). On day 14 of culture, the total cell number of mature HIO-derived InS^exp was ∼2.6 times greater compared to that of immature HIO-derived InS^exp (0.78±0.0265 (×10⁵) in immature HIO-derived InS^exp vs. 2.55±0.046 (×10⁵) in mature HIO-derived InS^exp) (Fig. 3B). Interestingly, CHIR 99021 treatment showed that mature HIO-derived InS^exp responded more sensitively to WNT pathway activation compared to immature HIO-derived InS^exp (Fig. 3A and 3B). Furthermore, the WNT target genes were confirmed to be highly expressed in mature HIO-derived InS^exp compared to that in immature HIO-derived InS^exp (Fig. 3C∼E). Activation of the canonical WNT pathway governs the expression of stemness- and proliferation-related genes (11, 12); the expression of these genes and proteins including CD44, β-catenin, ASCL2 and KI67 was also higher in mature HIO-derived InS^exp than that in immature HIO-derived InS^exp (Fig. 3C∼E). Our findings indicate that immature and mature HIO-derived InS^exp retain maturity-specific functional characteristics and gene expression profiles.