DOI: https://doi.org/10.21203/rs.3.rs-2122762/v1
Background:Physiologically relevant cell models, including organoids, are considered to be reliable tools for recapitulating human biology. Although organoids are useful for cell-based compound screening, which may facilitate drug development, their applications are limited. Intestinal organoids are composed of multiple types of intestinal epithelial cells found in vivoand replicate organ structures and complexities. A major limitation of using organoids in screening studies is the high cost of their culture when commercially available recombinant proteins are used.
Methods: We previously succeeded in reducing the cost of human intestinal organoid culture by using the conditioned medium (CM) of L cells that stably co-express Wnt3a, R-spondin1, and Noggin via lentiviral infection. Based on this, we worked on further cost reduction by replacing expensive materials with cheaper ones and expanded the organoids in a more cost-effective way for a large-scale assay.
Results: We replaced recombinant hepatocyte growth factor protein with CM for human intestinal organoid culture.Moreover, collagen gel was used instead of Matrigel for organoid culture, and organoid proliferation rate, as well as marker gene expression, was largely unchanged. The combination of these replacements significantly contributed to cost reduction for culturing organoids and organoid-oriented monolayer cells. Furthermore, compound screening of thousands of known bioactive substances was performed using human intestinal organoids cultured with the refined cost-reduction strategies, and several compounds with more selective cytotoxicity against organoid-derived cells than Caco-2 cells were identified. The mechanism of action of one of these compounds, YC-1, was further elucidated. We showed that YC-1 induces apoptosis through the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway.
Conclusions:These results indicate that our methodologies for cost reduction enable large-scale organoid culture and subsequent compound screening, which may further expand the application of intestinal organoids and organoids in general in various research fields, including both theoretical and applied science.
Phenotypic screening, which is based on cell phenotypes rather than molecular mechanisms, has contributed significantly to drug discovery (1). One of the key characteristics of this assay is the adoption of physiologically relevant cell models that accurately reflect the in vivo biology (2, 3). Although established cell lines have been used in the research fields of molecular biology, pharmacology, and compound screening, they do not necessarily represent conserved signaling pathways in native cells or tissues. In contrast, native cells, such as stem cell-derived cells and primary cells, may serve as a better physiological system than the conventionally used cell lines. However, they have limitations in terms of culture scalability due to the loss of proliferative capacity, accessibility to human tissues, and reproducibility of differentiation and/or maturation states.
Organoids are defined as three-dimensional structures derived from (pluripotent) stem cells, progenitor cells, and/or differentiated cells that self-organize through cell-cell and cell-matrix interactions to mimic the architecture and function of native tissues in vitro (4). They are expected to function as biological tools to replace animals and have industrial applications, such as regenerative medicine. Small intestinal organoids were the first established organoids with proliferative capacities in mice (5) and humans (6, 7). Previously, we have succeeded in improving the differentiation efficiency of human induced pluripotent stem (iPS) cells into small intestinal organoids with ileum-like properties (8, 9). Additionally, we established monolayers of intestinal epithelial cells (IECs) from human iPS cell-derived intestinal organoids (hiPSOs) (10) and primary human ileum organoids (hPIOs) (11). Although organoids differentiated from iPS cells are reported to be functionally less mature than organoids from adult tissues (12), we confirmed that several key functions specific to the intestinal tract, such as glucose transport, apolipoprotein B-48 secretion, and cytochrome P450 induction, which were not observed in colon adenocarcinoma-derived Caco-2 cells, were conserved in hiPSO-derived IECs (11), suggesting their usefulness in screening studies on intestinal function and metabolism. Since organoid culture requires expensive recombinant proteins, we previously developed a single line of mouse L cells simultaneously overexpressing Wnt3a, R-spondin1, and Noggin (L-WRN cells) by lentiviral infection and successfully reduced the culture cost by using the culture supernatant (conditioned medium, CM) as the organoid growth medium (8). However, further cost reduction is desired for assays that require large-scale cultures, such as screening studies.
Matrigel, a commercially available extracellular matrix (ECM) extracted from mouse sarcoma that contains many types of growth factors, has been routinely used for the culture of human organoids of the digestive system, including the stomach (13), intestine (6), liver (14), and pancreas (15). Despite its frequent use, it is accompanied by lot-to-lot variation and contains unknown humoral factors, which hinders human studies, including clinical trials (16).
In this study, we succeeded in reducing the culture cost of human intestinal organoids and organoid-derived IECs using CM from mouse L cells exogenously expressing hepatocyte growth factor (HGF), together with Wnt3a, R-spondin1, and Noggin (L-WRNH cells). In addition, we replaced Matrigel with type I collagen gel as an ECM to embed organoids for further cost reduction. We conducted compound screening with intestinal organoids cultured using a combination of these methods and discovered several compounds with selective cytotoxicity against organoid-derived IECs. In this work, we have established an intestinal organoid culture at a greatly reduced cost, which enables drug screening and make other intestinal organoid applications more feasible to be conduct, presumably leading to innovative studies that are impossible to conduct owing to budget issues, especially in academia.
Antibodies against cleaved caspase-3 (9661), caspase-8 (9746), poly (ADP-ribose) polymerase (PARP) (9542), and receptor-interacting protein 1 (RIP1) (3493) were purchased from Cell Signaling Technologies. Antibodies against mucin 2 (sc-7314), and X-linked inhibitor of apoptosis (XIAP) (sc-55551) were purchased from Santa Cruz Biotechnology. Antibodies against β-actin (A5441), Ki-67 (550609), and lysozyme (A0099) were purchased from Sigma-Aldrich, BD Biosciences, and Dako, respectively. The 4,6-diamidino-2-phenylindole (DAPI) solution was purchased from Cell Biolabs. Secondary antibodies for western blotting and immunostaining were purchased from Jackson ImmunoResearch, except for Alexa Fluor 568 conjugated anti-mouse IgG, which was from Thermo Fisher Scientific. The recombinant mouse epidermal growth factor (EGF) and human HGF were purchased from PeproTech and R&D Systems, respectively. A collagen gel culturing kit (638–00781) and type I-A collagen gel (631–00651 or 637–00653) were purchased from Nitta Gelatin (Japan). Native collagen acidic solution (IAC-50) and atelocollagen acidic solution (IPC-50) were purchased from Koken. Collagen gel reconstitution for 3D culture was performed according to the manufacturers’ instructions. Sodium alginate 80–120 was purchased from FUJIFILM Wako Chemicals. Alginate gel formation was conducted as per the previous study (17).
Caco-2 cells were cultured in Eagle’s minimum essential medium (MEM) supplemented with 10% FBS, 1× MEM non-essential amino acid solution, 100 units/ml penicillin, and 100 µg/mL streptomycin. For differentiation, Caco-2 cells were cultured in a monolayer for > 2 weeks after reaching confluence. The induction of stable transepithelial electrical resistance was confirmed before use in the assays.
L cells stably expressing human R-spondin1, human Noggin, and mouse Wnt3a with (L-WRNH) or without (L-WRN) human HGF were established by lentiviral infection, as previously described (8). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 µg/mL streptomycin. Each CM was prepared from supernatants seeded in a 1.4 × 106 cells/35 mm plate for 72 h.
The differentiation of human iPS cells (TkDN4-M) into intestinal organoids was performed as previously described (8). Human intestinal organoid (hiPSO and hPIO) culture and passage were as follows. Organoids embedded in Matrigel or collagen gel were washed with phosphate-buffered saline (PBS) and treated with either TrypLE Express solution (Thermo Fisher Scientific) for 5 min at 37℃ in a water bath without shaking (Matrigel) or 1 mg/mL collagenase solution for 15 min at 37℃ in a water bath with shaking (collagen gel). The organoids treated with collagenase were then collected by centrifugation at 440 × g for 3 min, washed twice with 10 ml of basal medium, and suspended in TrypLE Express solution. Subsequently, organoids were disrupted by vigorous pipetting 30–40 times and collected by centrifugation at 440 × g for 3 min. After removing the supernatant, the organoids were washed with 10 ml of basal medium (Advanced DMEM/F-12 supplemented with 10 mM HEPES (pH 7.3), 2 mM GlutaMAX, 100 units/mL penicillin, and 100 µg/mL streptomycin). After centrifugation at 440 × g for 3 min, the organoids were resuspended in Matrigel or reconstituted type I collagen gel with 20% growth medium [Advanced DMEM/F-12 with 25% WRNH CM (or 25% WRN CM) and 50 ng/mL human HGF), 10 mM HEPES (pH 7.3), 1% bovine serum albumin (BSA), 2 mM GlutaMAX, 50 ng/mL mouse EGF, 10 µM Y-27632, 10 µM SB202190, 500 nM A83-01, 100 µg/mL gentamicin, 100 units/mL penicillin, and 100 µg/mL streptomycin] on ice. Suspensions were aliquoted into the wells of Nunc 4-well plates (Thermo Fisher Scientific), ensuring that the border of each well was untouched, and solidified in a 5% CO2 incubator at 37°C for 15 min (Matrigel) or 30 min (collagen gel). Growth medium (500 µL) was then added to each well. The entire medium was changed every 3 days. Organoid passage was performed every 6 or 7 days. The passaging ratios ranged from 1:8 to 1:16. All the cultures were incubated in a 5% CO2 incubator at 37°C. The experiments using primary human ileum organoids (82-year-old female) complied with the Declaration of Helsinki and were approved by the human ethical committee of The University of Tokyo (No. 18–341) and Osaka University (No. 27-5-11). All tissues were sampled with informed consent.
After harvesting from the Matrigel or collagen gel, digestion, and disruption by pipetting 30 times in TrypLE Express solution, organoids were collected by centrifugation at 440 × g for 3 min. After washing with basal medium and harvesting by centrifugation, the cells were resuspended in growth medium, filtered through a 40-µm nylon mesh (Corning), and seeded in six-well plates coated with type I collagen or Transwells (all from Corning). The medium was replaced every 2–3 days. Cells were cultured in a 5% CO2 incubator at 37°C.
Total cellular RNA was extracted using RNeasy Mini Kit (Qiagen). Reverse transcription was performed using a high-capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). The mRNA levels were measured by fluorescence real-time PCR on StepOnePlus (Thermo Fisher Scientific) using PrimeTime qPCR assays (Integrated DNA Technologies). The 18S rRNA levels were used as an internal control to normalize the mRNA levels of each gene.
After the preparation of lysis buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and a protease inhibitor cocktail], cell lysates were subjected to SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. The membrane was blocked with Blocking One (Nacalai Tesque) for 1 h at room temperature (RT), incubated overnight at 4°C with primary antibodies against caspase-3 (1:1000), caspase-8 (1:1000), PARP (1:1000), and RIP1 (1:1000), and incubated with secondary antibodies for 1 h at RT. Chemiluminescent signals were determined using Fusion Solo S (Vilber).
Whole-mount immunofluorescence staining of organoids was performed according to a previous protocol (10). Briefly, organoids were fixed and permeabilized using a Cytofix/Cytoperm Kit (BD Biosciences). The specimens were then incubated with primary antibodies (rabbit anti-lysozyme (1:10), mouse anti-mucin 2 (1:50), or mouse anti-Ki67 (1:50)) at 4℃ overnight. After washing three times with Perm/Wash Buffer, organoid specimens were further incubated with Alexa Fluor 568-conjugated anti-mouse IgG (1:800) or Alexa Fluor 647-conjugated anti-mouse IgG (1:100) for 3 h at 4°C. After washing three times with Perm/Wash Buffer, the cells were incubated with DAPI (1:1000) for 10 min at RT. Fluorescence staining was visualized with fully focused z-stack images using an all-in-one fluorescence microscope (Keyence, Japan) equipped with structured illumination.
After harvesting from the Matrigel and disrupting by pipetting 30 times in TrypLE Express solution, organoids were collected by centrifugation at 440 × g for 3 min. After washing with basal medium and harvesting by centrifugation, the cells were resuspended in growth medium and filtered through a 40-µm nylon mesh, resulting in a dispersed state at the single-cell level. The cells were then seeded at 5.0 × 103 per well (96-well plate) or 1.2 × 103 per well (384-well plate) with or without the compounds and incubated for 48 h in a 5% CO2 incubator at 37°C. After CellTiter-Glo® 3D reagent (Promega) was added to each well and incubated for 10 min at RT, luminescence signals were measured using TriStar2 (Berthold) or PHERAstar (BMG Labtech), according to the manufacturers’ protocols.
Results are presented as the mean ± standard deviation (SD). Data were analyzed using Student's t-test for two groups. Differences were considered significant at P < 0.05 (indicated by asterisks).
In a previous study, we successfully established L cells that stably overexpressed Wnt3a, R-spondin1, and Noggin at each required concentration (8). The culture medium for human intestinal organoids was routinely prepared by adding 50 ng/mL human recombinant HGF to 4-fold diluted CM harvested from L-WRN cells, which enabled a proliferation rate similar to that of organoids cultured with WRN recombinant proteins. Although HGF is seemingly not essential for human intestinal organoid culture (6, 18), we previously found that it significantly increased organoid growth, as observed in primary human colon organoids (18), which would be beneficial for assays that require large numbers of organoids. Additionally, we showed that L-WRNH cells, whose culture supernatant contained approximately 200 ng/mL HGF, were established by infecting L-WRN cells with a human HGF lentivirus (8). However, we did not examine whether CM prepared from L-WRNH cells could be applied to human intestinal organoid culture for further cost reduction by replacing recombinant HGF in the culture medium.
We cultured hiPSOs with either 25% WRN or 25% WRNH CM and found that hiPSOs cultured with WRNH CM proliferated more prominently than those cultured with WRN CM, as determined by time-lapse microscopic observation (Fig. 1A) and viable cell numbers using CellTiter-Glo 3D reagents (Fig. 1B). Therefore, we compared the 25% WRNH CM with the conventional culture medium (25% WRN CM with 50 ng/mL recombinant HGF) from the perspective of hiPSO proliferation capacities. Time-lapse microscopy revealed that hiPSOs proliferated at similar rates in both media (Fig. 1C). Furthermore, we quantified the number of viable cells using CellTiter-Glo 3D reagents and found no difference between the two groups after 7 days of passage (Fig. 1D). Notably, CM prepared from intact L cells scarcely proliferated hiPSOs (Fig. 1A), showing that both FBS supplemented in the medium and endogenous secretions from intact L cells had little effect on organoid growth. Importantly, WRNH CM stored in a deep freezer for more than two years did not change the organoid proliferation capacity determined by time-lapse microscopic images (Fig. 1E) and viable cell numbers (Fig. 1F), indicating that WRNH CM can be stably stored for a long time. From these data, we conclude that WRNH CM is useful for expanding human intestinal organoids and contributes to their culture cost reduction.
In addition to changing the culture medium from WRN to WRNH CM, Matrigel was replaced with another ECM to embed organoids for 3D culture. Given that Matrigel is expensive and is derived from mouse sarcoma, which causes lot-to-lot variations and severely limits its use in clinical studies (19), an alternative candidate should be pure, xeno-free, and inexpensive. We selected type I collagen (18) and alginate hydrogels (17) as potential ECM for human intestinal organoid culture. Although the alginate hydrogel was much cheaper than the collagen gel, hiPSOs barely proliferated when the gel was used (Figure S1). Type I collagen gel for 3D culture is commercially available, and we chose three more inexpensive products than Matrigel, namely porcine tendon collagen (Nitta Gelatin), bovine dermis collagen (Koken), and bovine dermis atelocollagen (Koken), to compare their suitability for organoid culture. As observed in full-focused z-stack images, hiPSOs embedded in the porcine tendon collagen gel proliferated better than those embedded in the bovine dermis collagen gel (Figure S2). Atelocollagen is a low-immunogenic collagen derivative prepared by removing non-helical telopeptide regions of collagen by protease digestion (20). Although atelocollagen is somewhat cheaper than collagen, we found that the gel could not maintain stable dome structures during the culture and collapsed as organoids expanded (Figure S2), resulting in the loss of organoids before harvest. Therefore, we chose the type I collagen derived from the porcine tendon as a potentially suitable matrix for human intestinal organoid culture. As embedded in Matrigel, hiPSOs in the collagen gel proliferated more prominently with WRNH CM than with WRN CM (Figs. 2A and 2B), emphasizing the effectiveness of WRNH CM in the collagen scaffold. More importantly, time-lapse microscopy revealed that hiPSOs proliferated in the collagen gel at rates similar to those in Matrigel (Fig. 2C). Viable cell numbers determined by CellTiter-Glo 3D reagents were also found to be comparable between the two types of gel after 7 days of culture (Fig. 2D). Subsequently, we examined the mRNA levels of various intestinal epithelial genes using qRT-PCR. We found that the expression of the intestinal epithelial stem cell marker gene LGR5 (21), Paneth cell marker gene LYZ (6), goblet cell marker gene MUC2 (22), mature intestinal epithelial cell marker gene VIL1 (23), and intestinal gene that maintains IEC integrity HNF4A (24) was not significantly different between the organoids cultured with Matrigel and the collagen gel (Fig. 2E). Additionally, whole-mount immunostaining revealed that hiPSOs cultured with both Matrigel and the collagen gel contained proliferative cells (Ki67+), Paneth cells (lysozyme +), and goblet cells (mucin 2+) (Fig. 2F). Furthermore, we also confirmed the continuous growth of hiPSOs in the collagen gel over 10 passages with constant passage dilution rates. These results clearly indicate that the replacement of Matrigel with type I collagen gel did not significantly affect the organoid growth rate or fundamental intestinal marker gene expression, thus ensuring further cost reduction of organoid culture.
IECs can be developed from iPSOs and PIOs cultured with WRNH CM and collagen gel and can be maintained with WRNH CM
Despite their usefulness for examining physiological intestinal epithelium function and response, intestinal organoids may not be suitable for studies to evaluate the absorption and/or permeability of nutrients or drug candidates, especially those with high hydrophilicity, since the inside of the organoids corresponds to the intestinal lumen where digested food or xenobiotics physically interact. To overcome this issue, we previously established monolayer IECs from disrupted organoids by trypsinization, which allows exogenous factors to have direct access to the apical (luminal) side of the IECs (10, 25). Since the generation of monolayer IECs at low cost is considered to expand their applications in various assays, we attempted to develop IECs from hiPSOs cultured with WRNH CM and the type I collagen gel before propagating the IECs with WRNH CM. As a result, we confirmed the proliferation of hiPSO-derived IECs (Fig. 3A). Moreover, we previously showed that IECs can also be developed from hPIOs cultured with WRN CM (plus recombinant HGF) and Matrigel (11). We also confirmed the proliferation of hPIOs with WRNH CM and the collagen gel, as well as the proliferation of hPIO-derived IECs with WRNH CM (Fig. 3B). Furthermore, we examined whether WRNH CM-cultured IECs developed from hiPSOs propagated with collagen gel and WRNH CM exhibited characteristics similar to those of WRN CM plus recombinant HGF-cultured IECs developed from hiPSOs propagated with Matrigel, WRNH CM, and recombinant HGF. Gene expression analysis revealed that the mRNA levels of intestinal epithelial marker genes (LGR5, LYZ, MUC2, VIL1, and CDX2), as well as intestinal functional genes (HNF4A, MTTP, and CD36), were not statistically different between the IECs (Fig. 3C). These results indicate that physiologically relevant monolayer IECs can be developed from human intestinal organoids using WRNH CM and the type I collagen gel.
We recently performed compound screening and discovered that compounds with selective cytotoxicity against Caco-2 cells exclusively include anticancer drugs, highlighting that the origin of Caco-2 cells is cancer cells (11). To further investigate the different biological responses between intestinal organoids and Caco-2 cells, we performed a large-scale culture of hiPSOs with WRNH CM and the type I collagen gel and conducted another screening of compounds that selectively induce cytotoxicity against hiPSOs from a library of approximately 3500 chemical compounds consisting of drugs and pharmacologically active substances which were provided by Drug Discovery Initiative in the University of Tokyo. Consistent with a previous procedure, the dispersed IECs of organoids were used instead of organoids or monolayered IECs to promote cell homogeneity, which is important for cell-based drug screening to minimize measurement variation (26). Furthermore, SN-38, a topoisomerase I inhibitor and active metabolite of irinotecan, was chosen as a positive control compound that causes cytotoxicity against IECs because it is known to exhibit intestinal toxicity in vivo (27). The cytotoxic effect of SN-38 was confirmed by cell viability assay using CellTiter-Glo 3D Reagent, and 1 µM of SN-38 was used to guarantee the assay quality of each plate (Fig. 4A).
The cytotoxic compounds against organoid-derived IECs were first screened at every single concentration of 2 µM in 0.2% dimethyl sulfoxide. The average Z′-factor of the screening exceeded 0.6, indicating that the assay was sufficiently robust to filter the compounds (Fig. 4B). One hundred sixty-seven compounds with > 40% inhibitory activity, which was a statistical cutoff of 4×standard deviation (SD) of control groups without compound treatment (only with 0.2% dimethyl sulfoxide), were chosen as initial hits (Fig. 4C). Next, the hit compounds were evaluated by a counter-assay using Caco-2 cells, and 62 compounds with < 40% inhibitory activity were selected as potentially selective cytotoxic compounds against organoid-derived IECs (Figure S3). Subsequently, a dose-dependent analysis of these compounds was performed using Caco-2 cells and IECs derived from hiPSOs and hPIOs, and compounds with IEC-selective dose-dependent cytotoxicity were identified.
The identified compounds comprised ABT-737, N-(4-hydroxyphenyl)retinamide (4-HPR), ivermectin, N-oleoyldopamine, and 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1) (Fig. 4D). Although these compounds include antiproliferative agents, such as 4-HPR for breast cancer cells (28), ABT-737 for senescent cells (29), their effects on normal IECs have not been thoroughly investigated. These results revealed that drug responsiveness is significantly different between normal IECs and Caco-2 cells, which emphasizes the different physiological responses of intestinal organoids and Caco-2 cells.
YC-1 induces apoptosis through MEK (mitogen-activated protein kinase) /ERK (extracellular signal-regulated kinase) signaling in organoid-derived IEC
Among compounds with selective cytotoxicity against organoid-derived IEC, this study focused on YC-1, also called lificiguat, as this compound displayed least cytotoxicity against Caco-2 cells. YC-1 is known as an activator of soluble guanylyl cyclase (sGC) while, at the same time, an inhibitor of hypoxia-inducible factor-1α (HIF-1α) (30, 31). Previously, sGC activation was reported to inhibit human pulmonary arterial smooth muscle cell proliferation (32) and HIF-1α enhanced the proliferation of certain cells, including mesangial and renal carcinoma cells (33, 34). To elucidate whether the cytotoxic effect of YC-1 on IECs was mediated by sGC activation or HIF-1α inhibition, other chemotypes possessing the same actions as YC-1 namely, vericiguat as an sGC activator and LW6, PX-478, and PT-2385 as HIF-1α inhibitors, were used. Unexpectedly, these compounds failed to cause little cytotoxicity against IECs, suggesting that their cytotoxic effect on IECs was mediated by neither sGC nor HIF-1α (Fig. 5A and 5B). This conclusion is also supported by data showing that the expression of HIF1A and its target gene, VEGF, was comparable between monolayered IECs from hiPSOs and Caco-2 cells, regardless of their distinct sensitivity to YC-1 (Fig. 5C). Furthermore, we investigated whether YC-1 cytotoxicity was associated with the activation of apoptotic signals. YC-1 dose-dependently induced the cleavage of caspase-8 and its downstream pro-apoptotic proteins, RIP1, caspase-3, and PARP, together with decreased the anti-apoptotic protein, XIAP (Fig. 5D), indicating the induction of apoptosis. Consistent with a previous finding that YC-1-induced apoptosis accompanies the reduction of CCND1 expression (33), the mRNA levels of CCND1, as well as the levels of intestinal epithelial cell marker, VIL1, were found to be downregulated by the YC-1 treatment (Fig. 5E).
In previous studies, YC-1-induced apoptosis was mediated by the activation of the MEK/ERK (35), p38 MAPK (33), and Jun N-terminal kinase (JNK) (34) pathways in various cells. To further elucidate the pathway important for apoptosis induction in organoid-derived IECs, inhibitors of each kinase were used. Interestingly, while a p38 MAPK inhibitor (SB203580) and a JNK inhibitor (SP600125) had almost no effect, a MEK/ERK inhibitor (PD98059) substantially prevented the dose-dependent cytotoxicity induced by YC-1 (Fig. 6A, 6B, and 6C). Alternatively, PD98059 inhibited the cytotoxicity of 1 µM YC-1 in a dose-dependent manner (Fig. 6D). Finally, inhibition of YC-1 induced cytotoxicity by PD98059 was also confirmed in IECs from hiPSOs that were expanded with Matrigel (Fig. 6E), indicating that the difference of ECM composition had little effect on the cellular response to YC-1. Taken together, these results indicate that YC-1 caused cytotoxicity in organoid-derived IECs through the activation of MEK/ERK signaling.
The high physiology of cell models is considered essential for cell-based phenotypic screening, such as drug discovery. Recently, we reported that human organoid-derived IECs can perform crucial intestinal functions that cannot be replicated in Caco-2 cells (11), suggesting that the use of IECs would enable the evaluation of physiologically relevant in vivo events in a markedly precise manner. To increase their applications, we have tackled the issue caused by conventional organoid culture using commercially available recombinant proteins that incurs high costs and therefore requires cost reduction for routine or large-scale assays. To date, we have succeeded in reducing the cost by using CM harvested from L-WRN cells instead of recombinant proteins. In this study, we showed that further cost reduction could be achieved by replacing WRN CM and recombinant HGF with WRNH CM. Although the generation of WRN CM has been reported to date (36), which is independent of our lentiviral transduction system, the application of HGF-containing CM to organoid culture has not been reported. HGF is reported to enhance the proliferation of IECs (37) and can improve intestinal injury in vivo (38, 39), and has been used for human intestinal organoid culture (8, 18). In our organoid culture system, all recombinant proteins were successfully substituted with CM, except for EGF, which is an inexpensive cytokine.
Matrigel is known to be enriched in laminin, which is an effective ECM for organoid cultures (26, 40). However, because laminin is much more expensive than Matrigel, we believe that the use of laminin was not appropriate for the purposes of this study. A four-arm maleimide-terminated poly (ethylene glycol) macromer hydrogel is also a well-defined and appropriate matrix that may replace Matrigel for organoid culture (41). However, this material is more expensive than type I collagen gel. Collagen gel has been used for the culture of intestinal organoids (42, 43) but much less frequently than Matrigel. However, we demonstrate that human intestinal organoids can be cultured in type I collagen gel at a growth rate similar to that of Matrigel, and monolayer IECs can also be developed from organoids expanded with the collagen gel. To the best of our knowledge, this is the first study to represent the culture of human iPSC-derived normal intestinal organoids in a collagen scaffold. The price of type I collagen gel is approximately one-tenth of that of Matrigel, which can significantly contribute to experimental cost savings. In addition to cost reduction, type I collagen is physiologically relevant as a major ECM in the small intestine (44), and the use of collagen gel instead of Matrigel will be beneficial, especially for human studies, as it does not contain carcinogenic or unidentified humoral factors that may exert some unexpected effects. Notably, organoid-derived monolayer IECs would facilitate assays, such as absorption of bioactive molecules, intestinal barrier function, and co-culture with other cells, which may be difficult to conduct when using their parental organoids. Therefore, successful IEC culture at low costs can accelerate their use, leading to a more precise understanding of the physiological function of IECs, as well as the development of a new strategy for drug screening and development.
In this study, we proved that hiPSOs can be expanded on a large scale using WRNH CM and collagen type I gel. Through these extensive alterations, the cost associated with human intestinal organoid culture was reduced by up to 100-fold compared to initial costs. The resulting hiPSOs were used for the screening of thousands of compounds to identify those that selectively induce cellular toxicity against organoid-derived IECs. However, because the effects of these compounds on intestinal toxicity are currently unknown, there is a need to further investigate the extent to which IEC-selective compounds induce intestinal toxicity in humans. This study focused on YC-1, which exhibited significant cytotoxicity against IECs but very little against Caco-2 cells, and explored its mechanism of action. Considering that YC-1 is one of the HIF-1α inhibitors (31), and the intestinal tract is a hypoxic and anaerobic environment where HIF-1α can be induced (45), the toxicity exhibited by YC-1 against IEC was assumed to be associated with HIF-1α. However, other HIF-1α inhibitors with different mechanisms of action have little cytotoxicity effect. Furthermore, among several known pharmacological actions, YC-1 induces MEK/ERK-dependent apoptosis. Although the detailed molecular mechanism is currently unclear, the results demonstrate that intracellular signals are different between Caco-2 cells and normal IECs, suggesting the potential usefulness of IECs in screening studies or mechanism of action analysis.
Collectively, we successfully reduced the cost of culturing human intestinal organoids using WRNH CM and type I collagen gel. The method also enabled the culture of monolayered IECs developed from both hiPSOs and hPIOs. An increased number of compounds could be screened with organoids cultured using WRNH CM and type I collagen gel that helped identify compounds with higher cytotoxicity against IECs than against Caco-2 cells. These findings provide a basis for organoid research to accelerate a deeper understanding of the physiological relevance of human biology and industrial and clinical applications, including drug screening and transplantation.
Human intestinal organoids proliferated with L-WRNH CM and collagen gel, substituted by L-WRN CM and recombinant HGF and Matrigel, respectively, enabling the significant cost reduction of organoid culture. Screening of thousands of chemical compounds was attained using organoids expanded with the successive cost-reduction strategies, and YC-1 was identified that induced apoptosis selectively against the organoid-derived IECs through a MEK/ERK signaling pathway. Our methods could promote organoid research requiring large amounts of organoids, such as compound screening and regenerative medicine.
Bovine serum albumin
Conditioned medium
4,6-Diamidino-2-phenylindole
Dulbecco’s modified eagle’s medium
Extracellular matrix
Epidermal growth factor
Extracellular signal-regulated kinase
Fetal bovine serum
Hepatocyte growth factor
Hypoxia-inducible factor-1α
Human iPS cell-derived intestinal organoids
Primary human ileum organoids
N-(4-Hydroxyphenyl)retinamide
Intestinal epithelial cells
Induced-pluripotent stem
Jun N-terminal kinase
Mitogen-activated protein kinase
Minimum essential medium
Poly (ADP-ribose) polymerase
Phosphate-buffered saline
Receptor-interacting protein 1
Room temperature
Quantitative reverse transcription-polymerase chain reaction
Standard deviation
Wnt3a, R-spondin1, and Noggin
Wnt3a, R-spondin1, Noggin, and HGF
3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole
Ethics approval and consent to participate
The experiments using primary human ileum organoids complied with the Declaration of Helsinki and were approved by the human ethical committee of The University of Tokyo (No. 18-341) and Osaka University (No. 27-5-11). All tissues were sampled with informed consent.
Consent for publication
Not applicable.
Availability of data and materials
The datasets used during the current study are available from the corresponding author upon reasonable request.
Competing interests
The authors declare that they have no competing interests.
Funding
This study was supported by the Ministry of Education, Culture, Sports, Science, and Technology of Japan under Grant-in-Aid for Scientific Research A (20H00408 to R.S.); Grant-in-Aid for Scientific Research C (30835377 to Y.T.); Challenging Research (Exploratory, 22K18342 to R.S.); and the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research) from the Agency for Medical Research and Development (grant no. JP20am0101086; support no. 2472). The study was also supported by a joint grant from Kikkoman Corporation and by donations from the Hokuto Foundation for Bioscience.
Authors’ contributions
YT: Conception and design, financial support, collection and assembly of data, data analysis and interpretation, manuscript writing; YI: Collection of data; SS, TO, H.Kojima, H.Kiyono: Provision of study material, data analysis and interpretation; MS, YY: Administrative support; RS: Financial support, final approval of manuscript
Acknowledgments
We thank Dr. Otsu Makoto for providing human iPS cells and Dr. Riyo Imamura for the helpful discussions. We would like to thank Editage (www.editage.com) for English language editing.