You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

---

### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


Published: December 7, 2017

Introduction {#sec1}
============

Cell-based high-throughput screening for drug discovery is usually conducted using established cell lines or primary cells in 2D culture. However, current culture systems cannot satisfactorily recapitulate the complexity of the microenvironment and generally do not fully reflect cell phenotype *in vivo* ([@bib29]). Furthermore, evaluation of test compounds in animal models is not only costly but also carries uncertainty due to fundamental differences in physiology between humans and experimental animals. Indeed, pre-clinical animal studies cannot predict the toxicity of drug candidates in humans due to species differences ([@bib25], [@bib27]). Considering that species differences are also observed in the microtissues and cell clumps *in vitro* ([@bib13]), it is desirable to establish physiologically faithful 3D tissues from human cells as organ models.

Gut epithelial organoid culture is an emerging technique for investigating the molecular and cellular biology of the intestine *in vitro* ([@bib31], [@bib32], [@bib45]). Intestinal organoids are derived from intestinal epithelial stem cells (IESCs) and maintain self-propagation capacity because organoid crypt regions retain IESCs in addition to differentiated cells. WNT3A, R-spondin (RSPO) 1, and Noggin (NOG) are considered as key factors that enable self-proliferation of crypt IESCs. These organoids have been found to contain enterocytes, Paneth cells, goblet cells, and enteroendocrine cells derived from IESCs, as well as villus-like structures ([@bib31], [@bib33]). Thus, intestinal organoids possess many enteric characteristics found *in vivo*, and allow for greater use of human-derived culture systems in gut research, mitigating the uncertainty imposed by species differences.

Although epithelial organoids are powerful tools to investigate intestinal physiology and pathology, some cytokines essential for organoid growth are expensive if purchased commercially. Therefore, some attempts have been made to prepare conditioned medium (CM) for organoid culture from cells overexpressing specific cytokine genes ([@bib21], [@bib43]). However, stable cell lines usually express only one transgene, thereby requiring the production of multiple conditioned media from different cell lines, each overexpressing a single desired factor, or requiring several steps to transduce multiple genes in the same cell lines. Moreover, controlling their expression levels can usually be difficult. A retroviral or lentiviral expression system can overcome these disadvantages by enabling transduction of multiple genes simultaneously and efficiently in a single cell line, with control of expression levels by adjusting viral titers ([@bib4]). Usually, high titers can transduce more than one gene copy into 100% of exposed cells, eliminating the need for selection and cloning, and enabling establishment of stable cell lines in several days.

Matrigel, a proprietary extracellular matrix (ECM) gel, is often used as a scaffold material for 3D organoid culture ([@bib31], [@bib32]). However, use of Matrigel is unlikely to be appropriate for large-scale assays, such as drug screening, due to lot-by-lot variation in trace components and trial-by-trial variation in gel structure (porosity) ([@bib7]). For instance, Matrigel contains variable levels of growth factors such as fibroblast growth factor (FGF) 2, epidermal growth factor, and insulin-like growth factor 1 ([@bib40]). Furthermore, Matrigel is generated from mouse sarcoma and therefore may contain carcinogenic factors. It is thus not well suited for human studies, including clinical trials ([@bib15]). Some investigators have used collagen type I gel instead of Matrigel for organoid culture to avoid such concerns ([@bib10], [@bib45]); however, both collagen I gel and Matrigel require complicated procedures to recover cells for collection. Therefore, alternative methods for culture of human organoids are required.

Induced pluripotent stem cells (iPSCs), under appropriate conditions, can differentiate into multiple cell types, including cardiomyocytes, neurons, hepatocytes, and pancreatic cells ([@bib11], [@bib35], [@bib46], [@bib47]), making them invaluable for understanding embryonic development, drug toxicity testing, individualized research, and regenerative medicine ([@bib9], [@bib37]). Furthermore, iPSCs can be more readily obtained than embryonic stem cells, and their isolation carries fewer ethical concerns ([@bib23]). More importantly, use of human iPSCs from patients could lead to the development of disease-specific human drug-screening models, even when the cause of the disease is unclear. It has been reported that intestinal organoids with specific properties (e.g., crypt-villus axis) can be generated from human iPSCs ([@bib19]). Although iPSC-derived organoids are useful for studies on intestinal development and *in vivo* transplantation ([@bib42]), their application in high-throughput screening remains difficult due to limited culture scalability. In addition, stable gene transduction in human organoids can be precious, especially for regenerative medicine and drug screening. Although some researchers reported successful gene transduction in human intestinal organoids ([@bib5], [@bib36]), an easier and more rapid approach to modify the genes of interest is desired.

In this study, we developed or improved a number of methods to handle iPSC-derived intestinal organoids easily. First, we adopted lentiviral vector to readily establish and modify CM for human intestinal organoid culture. Second, we differentiated human iPSCs into intestinal organoids more efficiently by supplementing WNT3A and FGF2 during the differentiation into definitive endoderm (DE). Third, we were able to transduce an exogenous gene efficiently into these organoids through 2D culture. Fourth, we successfully cultured human iPSC-derived organoids in Happy Cell Advanced Suspension Medium (ASM), which does not require Matrigel and enables organoids to be easily collected. The combination of these techniques enables more efficient intestinal organoid culture and provides a scalable strategy to produce large numbers of organoids for therapeutic drug screening.

Results {#sec2}
=======

Preparation of CM for Organoid Culture {#sec2.1}
--------------------------------------

We first established a cell line that can stably express the cytokines WNT3A, RSPO1, and NOG, to reduce costs and labor for the development and maintenance of human organoids. Although a cell line simultaneously expressing mouse WNT3A (mWNT3A), mouse RSPO3, and mouse NOG has already been established and deposited to the American Type Culture Collection ([@bib21]), it was originally developed for using mouse organoid culture ([@bib20]). Therefore, we selected a lentiviral expression system for rapid establishment of our original cell line and characterized the CM produced by these cells. Prior to the establishment of such cells, we unexpectedly found that recombinant mWNT3A exhibited higher activity than recombinant human WNT3A (hWNT3A), as measured by a luciferase assay using a TOPflash reporter gene plasmid, which can detect Wnt signal activation ([@bib12]) ([Figure 1](#fig1){ref-type="fig"}A). In contrast to WNT3A, RSPO1 activities, which enhance Wnt signals, were comparable between mice and humans ([Figure 1](#fig1){ref-type="fig"}B). Therefore, we chose mWNT3A, human RSPO1 (hRSPO1), and human NOG (hNOG) (WRN) as the ingredients of CM for human organoid culture. Moreover, we compared Wnt activities of culture supernatants among three host cells, HEK293T cells, Chinese hamster ovary cells, and L cells, all of which were similarly infected with lentiviral vectors for WRN expression. Results indicated that L cells had the highest capacity to secrete WRN proteins among the three cell types (data not shown); therefore, we chose L cells as hosts to prepare WRN CM.Figure 1Preparation of WRN CM by Lentiviral Infection(A and B) Wnt activities of recombinant hWNT3A or mWNT3A (A) and recombinant hRSPO1 or mRSPO1 (B). Activities in the absence of the hRSPO1 expression plasmid and recombinant proteins were considered as "1." The assays were performed in three independent biological replicates. Data expressed as mean ± SEM. n.s., not significant (Student's t test).(C--H) Mouse L cells stably expressing WRN were generated by lentiviral infection of each gene. (C--E) Wnt signaling activities of CM from L-WRN cells at densities of 2.8, 1.4, 0.7, and 0.35 × 10^6^ cells/35-mm dish after 48 hr of culture (C); at a density of 1.4 × 10^6^ cells/dish cultured between 24 and 96 hr (D); and at 1.4 × 10^6^ cells/dish cultured for 72 hr in diluted CM or 300 ng/mL recombinant mWNT3A, with or without an hRSPO1 expression plasmid (E). Activities in the absence of WRN CM were considered as "1." The assays were performed in three independent biological replicates. Data expressed as mean ± SEM. (F) Western blot analysis of the supernatants used in (C) undiluted (8 μL) and 4-fold diluted, and the recombinant proteins mWNT3A (1 and 0.3 ng), hRSPO1 (30 and 10 ng), and hNOG (10 and 3 ng) using anti-WNT3A, anti-RSPO1, and anti-NOG antibodies. (G) Images of human primary ileal organoids cultured in 25% WRN CM or the corresponding recombinant proteins for 7 days after a passage. Scale bars, 200 μm. (H) Wnt signaling activities of WRN CM (×1, ×1/3, ×1/10) stored in the deep freezer for 0, 6, or 12 months. The activities in the absence of CM were considered as "1." The assay was performed in three independent biological replicates. Data expressed as mean ± SEM.

To optimize the conditions for preparing WRN CM, we seeded WRN-expressing L cells in 35-mm dishes at densities ranging from 0.35 × 10^6^ to 2.8 × 10^6^ cells in 2 mL medium, and harvested the medium 48 hr later. We found that a density of 1.4 × 10^6^ cells/dish yielded CM the maximum Wnt signaling activity ([Figure 1](#fig1){ref-type="fig"}C). We then examined the appropriate culture period by seeding WRN-expressing L cells at 1.4 × 10^6^ cells/35-mm dish and culturing them for 24--96 hr, which showed that the activity peaked at 72 hr ([Figure 1](#fig1){ref-type="fig"}D). Using these conditions, we found that WRN CM activity decreased with dilution and that undiluted WRN CM had higher activity than 300 ng/mL recombinant mWNT3A, even in hRSPO1 overexpressing cells ([Figure 1](#fig1){ref-type="fig"}E). We then examined the expression of each cytokine in WRN CM by western blot analysis ([Figure 1](#fig1){ref-type="fig"}F), and estimated that the concentrations of mWNT3A, hRSPO1, and hNOG were approximately 400 ng/mL, 3 μg/mL, and 800 ng/mL, respectively. Based on previous reports ([@bib8], [@bib32]), we assumed that 25% WRN CM would contain sufficient concentrations of these factors for human organoid culture. In fact, human primary small intestinal organoids could be cultured with medium containing 25% WRN CM as a source of required cytokines ([Figure 1](#fig1){ref-type="fig"}G). Importantly, the biological activities of WRN CM did not change even after 1 year of storage at −80°C ([Figure 1](#fig1){ref-type="fig"}H), which is an asset for the consistency of human organoid culture.

Differentiation of Human iPSCs into Intestinal Organoids {#sec2.2}
--------------------------------------------------------

We attempted to establish human intestinal organoids from iPSCs as a first step toward developing healthy and disease models for future studies. We first differentiated the human iPSC line TkDN4-M ([@bib39]) into DE using activin A for 3 days, and then treated the cells with FGF4 and WNT3A for up to 4 days to obtain spheroids according to previous reports ([@bib19], [@bib36]) ([Figure 2](#fig2){ref-type="fig"}A). However, the generation efficiencies of the spheroid- and subsequent organoid-like cell clumps were not sufficient ([Figure 2](#fig2){ref-type="fig"}B, left panels), and we were unable to improve this even by use of another human iPSC line, TkDA3-4 ([@bib39]) (data not shown). We speculated that this limitation was caused by low differentiation efficiency into DE. Therefore, we also added WNT3A and FGF2 during the initial culture stage, as these factors have been reported to enhance endoderm differentiation ([@bib18], [@bib30], [@bib34]) ([Figure 2](#fig2){ref-type="fig"}A). This modification greatly improved spheroid-like clump formation efficiencies ([Figures 2](#fig2){ref-type="fig"}B, right upper panel, and 2D), which was consistent with the upregulation of DE markers *sex determining region Y-box 17* (*SOX17*) and *C-X-C chemokine receptor type 4* (*CXCR4*) expression ([Figure 2](#fig2){ref-type="fig"}E), and further improved the following organoid-like clump formation efficiencies ([Figure 2](#fig2){ref-type="fig"}B, right lower panels).Figure 2Differentiation of TkDN4-M into Human Intestinal Organoids(A) Schematic procedures for intestinal organoid differentiation from human iPSC lines.(B) Images of the human iPSC line TkDN4-M that had been differentiated into spheroids (upper panels) and organoids (lower panels) with (right panels) or without (left panels) recombinant hWNT3A and human FGF2 (hFGF2). Arrowheads indicate spheroid- and organoid-like cell clumps. Scale bars, 200 μm.(C) Magnified images of the spheroids and organoids. Scale bars, 40 μm.(D) The numbers of spheroids per microscopic bright field in the presence or absence of WNT3A/FGF2 supplementation. The assays were performed in three independent experiments. Data are presented as mean ± SEM. ^∗^p \< 0.05, Student\'s t test.(E) Relative mRNA levels of SOX17 and CXCR4 in TkDN4-M cells differentiated into spheroids in the presence or absence of WNT3A/FGF2 supplementation were determined by qPCR and normalized to HPRT. The assay was performed in three independent biological replicates. Expression levels presented as mean ± SEM. ^∗^p \< 0.05, Student\'s t test.(F) Relative mRNA levels of the indicated genes in TkDN4-M cells during the course of differentiation (days 0, 3, and 7 \[spheroids\], and organoids) were determined by qPCR and normalized to *HPRT*. The highest expression levels of each gene in each differentiation stage were considered as "1." The assays were performed in three independent biological replicates. Expression levels presented as mean ± SEM.(G) Images of TkDN4-M-derived organoids on day 7 after culture in 25% WRN CM, or the corresponding recombinant proteins. Scale bars, 200 μm.(H) Relative mRNA levels of *LGR5* and *VIL1* in TkDN4-M organoids cultured for short-term (passage 19) and long-term (passage 53) were determined by qPCR and normalized to *HPRT*. The assays were performed in three independent biological replicates. Expression levels presented as mean ± SEM.(I) Whole-mount images of TkDN4-M-derived organoids stained with phalloidin (blue), together with each indicated antibody. Scale bars, 50 μm.

The morphologies of these clumps were similar to those reported previously for spheroids and organoids ([Figure 2](#fig2){ref-type="fig"}C) ([@bib19]). Furthermore, the relative gene expression of the endoderm markers *SOX17*, *CXCR4*, and *forkhead box protein A2* (*FOXA2*), peaked when the spheroid-like clumps were formed, whereas the intestinal epithelial markers *caudal-related homeobox transcription factor 2* (*CDX2*), *glycoprotein A33* (*GPA33*), and *villin 1* (*VIL1*), as well as the Paneth cell marker *lysozyme* (*LYZ*), were induced during organoid-like clump formation ([Figure 2](#fig2){ref-type="fig"}F). Thus, these clumps clearly possessed the typical properties as spheroids and organoids, demonstrating that terminal differentiation into intestinal organoids proceeded successfully. We also confirmed that the organoids were able to proliferate in our 25% WRN CM, as well as in recombinant proteins such as the organoids prepared from ileum ([Figures 2](#fig2){ref-type="fig"}G and [1](#fig1){ref-type="fig"}G). Furthermore, expression of *LGR5* and *VIL1* was comparable between short-term (100 days) and long-term (300 days) cultures, indicating that properties of human iPSC-derived organoids are stable over multiple passages ([Figure 2](#fig2){ref-type="fig"}H). Finally, whole-mount staining revealed the development of Paneth cells (LYZ^+^), goblet cells (MUC2^+^), enteroendocrine cells (Chromogranin A \[CHGA\]^+^), and proliferative cells (MKI67^+^), confirming successful organoid differentiation from TkDN4-M ([Figure 2](#fig2){ref-type="fig"}I).

We were also able to obtain organoid-like cell clumps from other human iPSC lines, TkDA3-4 ([Figure S1](#mmc1){ref-type="supplementary-material"}A) and TkCBSev9 ([@bib44]) ([Figure S1](#mmc1){ref-type="supplementary-material"}B), by the addition of WNT3A and FGF2 to the differentiation medium, and similarly confirmed their authentic organoid properties through extensive gene expression analysis ([Figures S1](#mmc1){ref-type="supplementary-material"}C and S1D). No organoids were obtained from TkCBSev9 without the addition of WNT3A and FGF2 during the initial stage of the differentiation (data not shown). These results indicate that our modified differentiation protocol, which enables higher differentiation capacities into intestinal organoids, can be generally applied.

Comparison between Human iPSC-Derived Organoids and Primary Organoids from the Small Intestine and Colon {#sec2.3}
--------------------------------------------------------------------------------------------------------

Spence et al. have reported, using transcriptome analysis, that intestinal organoids differentiated from human iPSCs show mid- and hindgut specification ([@bib19]) and possess small intestinal properties ([@bib3]). To investigate whether our intestinal organoids, which were differentiated by the modified method, show small intestinal specifications, we prepared human primary organoids from healthy parts of the ileum and transverse colon of surgical specimens using human organoid culture medium containing 25% WRN CM, and found that both types of primary organoids showed an almost identical morphology to human iPSC-derived organoids ([Figure 3](#fig3){ref-type="fig"}A).Figure 3Comparison of Human iPSC-Derived Organoids with Primary Intestinal Organoids(A) Images of organoids from the human primary ileum or transverse colon cultured with human organoid culture medium for 6 days. Scale bars, 200 μm.(B) Relative mRNA levels of the indicated genes in organoids derived from human primary ileum, colon, and iPSCs harvested at 6 days after passage. The relative mRNA levels were determined by qPCR and normalized to *GAPDH*. The assays were performed in three independent biological replicates. Data are presented as mean ± SEM.

Based on a recent DNA microarray that compared the gene expression profiles of different parts of the human intestinal epithelium ([@bib41]), we chose to examine the expression of the *apolipoprotein* (*APO*) *B* and *APOC3* genes, and found that their mRNA levels were much higher in organoids from the small intestine and iPSCs than in organoids from the colon ([Figure 3](#fig3){ref-type="fig"}B). We also examined the expressions of *carcinoembryonic antigen-related cell adhesion molecule 7* (*CEACAM7*) and *MUC2*, which were higher in organoids from the colon than in those from the small intestine and iPSCs. As a control gene, we examined *nucleotide-binding oligomerization domain containing 1* (*NOD1*), and confirmed that its expression, as well as that of *VIL1*, was comparable across the three types of organoids examined. These results indicate that characteristics of our human iPSC-derived organoids are much closer to those of small intestinal organoids than colonic organoids.

Gene Transduction into Human iPSC-Derived Organoids by Lentiviral Infection {#sec2.4}
---------------------------------------------------------------------------

Stable gene transduction into human iPSC-derived organoids would be required to create organoids that mimic certain diseases or to screen for possible therapeutic genes. However, since organoids self-proliferate as cell clumps, and are usually seeded in solid ECM such as Matrigel, it is difficult to simply apply typical transfection and selection methods for stable gene expression. At first, in reference to a previous report to transduce exogenous genes into human iPSC-derived organoids using adenoviruses ([@bib36]), we recovered organoids from Matrigel and incubated them with lentiviruses for Venus expression; however, the transduction efficiencies as well as Venus expression intensities were not sufficiently high ([Figures 4](#fig4){ref-type="fig"}B and 4C; 3D groups). Therefore, we devised a unique strategy to transduce a gene into human iPSC-derived organoids. We have found that when organoids were physically disrupted using needles and seeded on collagen I-coated dishes, 2D-proliferative cells emerged ([Figure 4](#fig4){ref-type="fig"}A). Intriguingly, when these cells were harvested and re-embedded in Matrigel, organoids were again generated, indicating that the cultures containing sufficient numbers of IESCs can reconstitute organoids in Matrigel ([Figure 4](#fig4){ref-type="fig"}A). Considering these results, we postulated that gene transduction into iPSC-derived organoids can be achieved by organoid disruption, followed by lentiviral spin infection of 2D-cultured IESCs and re-embedded them in Matrigel. Based on this notion, we examined lentiviral infection efficiency in organoids by monitoring Venus expression, and found that more than 70% of organoids were transduced ([Figures 4](#fig4){ref-type="fig"}B and 4C; 2D groups). We observed no significant difference in regenerated organoid yield at 1 week between infected and non-infected groups ([Figure 4](#fig4){ref-type="fig"}D), demonstrating that lentiviral infection had little effect on IESC viability. Last, to assess exogenous gene function in organoids, we introduced *Wnt3a*-internal ribosome entry site 2 (IRES2)-Venus plasmid into organoids using the above strategy, and found that they stably expressed mWNT3A and Venus even after several passages ([Figures 4](#fig4){ref-type="fig"}E and 4F). Furthermore, we found that the expression of Wnt target genes, namely *LGR5*, *AXIN2*, and *EPHB3*, was induced by exogenous mWNT3A ([Figure 4](#fig4){ref-type="fig"}F), suggesting that functional Wnt signaling was stably enhanced.Figure 4Lentiviral Gene Transduction into Human iPSC-Derived Intestinal Organoids(A) Schematic representation of gene transduction into organoids. TkDN4-M-derived organoids cultured in Matrigel were disrupted by a 29G needle, seeded in collagen I-coated plates, and cultured for 4 days. After 2D cultures from organoids were infected by lentiviruses, cells (including IESCs) were trypsinized, harvested, and re-embedded in Matrigel to reform organoids.(B) The proportions of Venus^+^ organoids per microscopic bright field after 7 days of infection with IRES-Venus lentivirus. "3D" and "2D" indicate lentiviral infection into organoids harvested from Matrigel and 2D-cultured cells in collagen I-coated plates, respectively. The assay was performed in three independent biological replicates. Data are presented as mean ± SEM. ^∗^p \< 0.05, Student\'s t test.(C) Bright-field and fluorescent images of TkDN4-M-derived organoids as in (B). Scale bars, 200 μm.(D) Regenerated organoid numbers per microscopic bright field followed by 2D cultures after 7 days of infection with or without IRES-Venus lentivirus. The assay was performed in three independent biological replicates. Data are presented as mean ± SEM.(E) Bright-field and fluorescent images of TkDN4-M-derived organoids 3 weeks after infection with *Wnt3a*-IRES-Venus lentivirus in 2D cultures. Scale bars, 200 μm.(F) Relative mRNA levels of exogenous *Wnt3a* and Wnt target genes (*LGR5*, *AXIN2*, and *EPHB3*) in TkDN4-M-derived organoids infected with lentiviruses as in (E). The relative mRNA levels were determined by qPCR and normalized to *GAPDH*. The assay was performed in three independent biological replicates. Expression levels presented as mean ± SEM. ^∗^p \< 0.05, Student\'s t test. n.d., not detected.

Characterization of Human iPSC-Derived Organoids Cultured in Suspension Medium {#sec2.5}
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Although 3D culture in a solidified environment, such as Matrigel, is a well-established method for culturing intestinal organoids ([@bib32]), it is often laborious and inappropriate for large volume culture because the gel impedes cell recovery. To overcome this problem, we tried to maintain organoids in suspension culture without solid ECM. As suspension medium, we adopted Happy Cell ASM (Biocroi, Ireland) ([@bib14]) and FCeM (Nissan Chemical Industries) ([@bib26]), both of which are commercially available but mainly used for cancer spheroid culture. We then prepared a suspension medium supplemented with 25% WRN CM, and cultured TkDN4-M-derived organoids in low-attachment plates. Time-lapse microscopy revealed that the organoids were able to grow in Happy Cell ASM as well as in Matrigel; however, they failed to grow in FCeM ([Figure 5](#fig5){ref-type="fig"}A). We confirmed that the organoids grown in Happy Cell ASM contained Paneth cells, goblet cells, and enteroendocrine cells ([Figure 5](#fig5){ref-type="fig"}B), as in Matrigel ([Figure 2](#fig2){ref-type="fig"}I). Furthermore, we stimulated the organoids with interleukin-22 (IL-22), a potent inducer of epithelial cell cytokine expression and regeneration ([@bib17]), to examine physiological responsiveness. *Regenerating family member 3α* (*REG3A*) expression was dose-dependently induced by IL-22 in organoids cultured in both Matrigel ([Figure 5](#fig5){ref-type="fig"}C) and Happy Cell ASM ([Figure 5](#fig5){ref-type="fig"}D), consistent with the IL-22 response in mouse intestinal organoids ([@bib17]).Figure 5Growth of Human iPSC-Derived Organoids by Suspension Culture Using Happy Cell ASM(A) Time-lapse bright-field images of TkDN4-M-derived organoids cultured in Matrigel, Happy Cell ASM, or FCeM, each supplemented with 25% WRN CM and requisite ingredients for human organoid culture. Scale bars, 200 μm.(B) Whole-mount images of TkDN4-M-derived organoids cultured in Happy Cell culture medium were stained with phalloidin (blue), together with each indicated antibody. Scale bars, 50 μm.(C and D) Relative mRNA levels of the indicated genes in TkDN4-M-derived organoids cultured in Matrigel (C) or Happy Cell ASM (D) for 5 days and then exposed to each pre-stimulation medium containing the indicated concentrations of recombinant human IL-22 for 2 days. The relative mRNA levels were determined by qPCR and normalized to *GAPDH*. The assays were performed in three independent biological replicates. Data presented as mean ± SEM.

We next conducted serial PCR analyses and found that expression of the stem cell markers *LGR5* and *ASCL2* was lower in whole-mount organoids cultured in Happy Cell ASM than those in Matrigel, whereas expression levels of *VIL1* and the goblet marker *MUC2* were higher ([Figure 6](#fig6){ref-type="fig"}A), indicating that organoids cultured in Happy Cell ASM are more likely to be composed of differentiated IECs, rather than IESCs. Based on these results, we performed DNA microarray analysis to comprehensively compare gene expression changes caused by suspension culture after two passages. Representative genes showing up- and downregulation are presented in [Tables S1](#mmc2){ref-type="supplementary-material"} and [S2](#mmc3){ref-type="supplementary-material"}. Among these genes, the expression of another intestinal epithelial marker, *GPA33*, was increased when organoids were cultured in Happy Cell ASM ([Figure 6](#fig6){ref-type="fig"}A), further indicating that suspension culture increased certain populations of differentiated IECs. In contrast, when recombinant mWNT3A was added to the medium, *LGR5* and *ASCL2* expression was induced and *VIL1* and *MUC2* expression was decreased ([Figure 6](#fig6){ref-type="fig"}B), which suggests that proportions of cell components can be modified by changing medium ingredients. This notion was also supported by whole-mount immunostaining against MKI67, which indicates that MKI67^+^ proliferative cells were increased by WNT3A supplementation in the organoids cultured in Happy Cell ASM ([Figure 6](#fig6){ref-type="fig"}C).Figure 6Characterization of Human iPSC-Derived Organoids Cultured in Happy Cell ASM(A) Relative mRNA levels of the indicated genes in TkDN4-M-derived organoids cultured with Matrigel (open bars) or Happy Cell ASM (closed bars) plus 25% WRN CM and requisite ingredients for human organoid culture after one (P1) or two (P2) passages in each culture condition. The relative mRNA levels were determined by qPCR and normalized to *GAPDH*. The assays were performed in three independent biological replicates. Data presented as mean ± SEM. ^∗^p \< 0.05, Student\'s t test.(B) Relative mRNA levels of the indicated genes in TkDN4-M-derived organoids cultured in Happy Cell ASM with or without 300 ng/mL recombinant mWNT3A for 6 days. The relative mRNA levels were determined by qPCR and normalized to *GAPDH*. The assays were performed in three independent biological replicates. Data presented as mean ± SEM. ^∗^p \< 0.05, Student\'s t test.(C) Whole-mount images of TkDN4-M-derived organoids cultured in Happy Cell ASM with or without 300 ng/mL recombinant mWNT3A after staining with phalloidin (blue) and anti-MKI67 antibodies. Scale bars, 50 μm.

Discussion {#sec3}
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Intestinal organoids derived from IESCs contain various IEC types observed *in vivo*, including enterocytes, Paneth cells, goblet cells, and enteroendocrine cells; they are, thus, regarded as "mini-organs" ([@bib2]). Unlike normal cell lines or primary cultures, however, organoid culture is both expensive and labor intensive. Moreover, it is difficult to establish organoids stably expressing exogenous genes for research applications, particularly for studies requiring disease-mimicking organoids. In this study, we developed several original protocols and modified others to improve the efficiency, cost, and practicality of human iPSC-derived organoid culture. First, we produced a single line of L cells stably co-expressing mWNT3A, hRSPO1, and hNOG, factors required for IESC maintenance, by lentiviral infection. Importantly, unlike transfection methods (e.g., lipofection and electroporation), lentivirus infection enables not only transduction of many genes into the same cell genome promptly and simultaneously, but also control of expression levels by changing viral titers. Indeed, we were able to generate WRN CM for human organoid culture with desired and reproducible concentrations of mWNT3A, hRSPO1, and hNOG by adjusting the titers of each virus (*Wnt3a*:*RSPO1*:*NOG* = 15:3:1). Furthermore, it should also be possible to easily prepare other CM types by establishing L cells co-expressing different combinations of genes by lentiviral infection. As substantial examples, we further generated human hepatocyte growth factor (HGF)- and/or IL-22-producing L-WRN cells by lentiviral infection because these cytokines have the capacity to stimulate organoid growth ([@bib17], [@bib45]). We found that L-WRN cells infected with both *HGF*- and *IL22*-expressing lentiviruses can simultaneously produce CM containing approximately 200 ng/mL of HGF and 80 ng/mL of IL-22 ([Figure S2](#mmc1){ref-type="supplementary-material"}A). We used the CM for human iPSC-derived organoid culture at 25% concentration as normal, and confirmed that HGF- and IL-22-containing WRN CM cooperatively increased organoid growth, which was revealed by bright-field microscopic observation ([Figure S2](#mmc1){ref-type="supplementary-material"}B) and quantification of viable cell numbers ([Figure S2](#mmc1){ref-type="supplementary-material"}C). Therefore, our strategy can help researchers in various fields to readily develop original CM-producing cells stably expressing any gene of interest with a variable expression level. The customized CM could greatly reduce cost and labor for human organoid culture by obviating the need for commercial factors without sacrificing inter-trial reproducibilities.

It is widely known that iPSC differentiation capacity varies among iPSC lines ([@bib24]), and we found that several iPSC lines, including TkDN4-M and TkDA3-4, failed to generate substantial numbers of intestinal organoids using a previous culture method ([@bib19]). Therefore, we improved the conditions for differentiation of human iPSCs into intestinal organoids by the addition of WNT3A and FGF2 during the initial culture phase. Efficient yield of the organoids from human iPSCs mitigates not only the ethical concerns of using fetal tissue but also the low availability of surgical specimens. In addition, it is expected that intestinal organoids can be generated even from iPSC lines with relatively low differentiation capacities by adopting the modified conditions used in our study. In fact, we were able to obtain any organoids from TkCBSev9 only when supplemented with WNT3A and FGF2.

Furthermore, we developed a method to stably express exogenous genes in organoids involving disruption, seeding in 2D culture for lentiviral infection, and subsequent organoid regeneration. It is worth mentioning that many of IESCs can survive and grow in 2D conditions because the number of regenerated organoids after re-embedding in Matrigel was comparable with that of organoids that were not subjected to 2D cultures. The 2D-cultured cells can be treated like normal adherent cultured cells with a proliferation capacity, which makes it easier to perform preliminary experiments such as determination of the MOI of multiple viruses simultaneously. The regeneration can be completed in a week, which is sufficient time for subsequent assays, and the overall method is easy and simple, and usually requires less than 2 weeks to obtain genetically engineered organoids. These properties suggest that our culture method is useful for patient-specific studies and production of organoids with disease-specific genetic abnormalities, and also be applicable to modern gene-editing strategies, such as the CRISPR/Cas9 system ([@bib28]). Moreover, we found that transient gene transduction by lipofection was also successfully achieved in 2D-cultured cells, and the expression of the exogenous gene was confirmed in regenerated organoids ([Figure S3](#mmc1){ref-type="supplementary-material"}), suggesting that lipofection-mediated gene transduction into organoids is performable in our system.

Gene expression analysis indicated that our human iPSC-derived organoids resembled those derived from the small intestine rather than the colon. Considering that the expression of *APOB*, *APOC3*, and *VIL1*, exclusively expressed genes in the small intestine, was comparable between iPSC-derived and primary organoids, it is plausible that the iPSC-derived organoids would mainly comprise cells that terminally differentiated toward the intestinal lineage. Therefore, they could be used to improve our molecular and cellular understanding of physiological and inflammatory conditions of the small intestine. In turn, such studies may facilitate development of new treatment alternatives to surgical excision of the small intestine, which has a much more severe impact on quality of life than excision of the colon. On the other hand, since previous reports have proposed that intestinal regiospecificities depend on the intrinsic gene expression profiles of local stem cells rather than differences in microenvironment ([@bib6]), it is desirable to also establish a method for generating human colon organoids from iPSCs. During the preparation of this manuscript, successful differentiation into colonic organoids from human iPSCs was reported by independent groups ([@bib1], [@bib22]). The differentiation protocols, particularly after CDX2^+^ spheroid formation, are different from the previous protocol that was used to obtain small intestinal organoids; therefore, there would be little chance of a contamination of colonic cells in the intestinal organoids differentiated using our method, which is a modified method of the original one to provide small intestinal properties.

Matrigel is a widely used ECM gel for organoid culture, but it is still cumbersome to handle because it must be dissolved before the recovery of organoids. In this study, we found that human iPSC-derived organoids self-propagated in suspension culture with Happy Cell ASM. Importantly, primary human ileal and colonic organoids could also proliferate in the suspension culture ([Figure S4](#mmc1){ref-type="supplementary-material"}). Although we have not yet identified the factors in Happy Cell ASM contributing to growth (which are presumably absent in FCeM), suspension culture in Happy Cell ASM can save time and labor for seeding and recovery of organoids, especially when a large number of organoids are needed. However, organoids expanded in Happy Cell ASM showed differences in cell marker expression compared with organoids in Matrigel, presumably due to ECM and trace growth factors in Matrigel ([@bib40]), which may allow IESCs to grow more efficiently. Consistent with this, organoid growth rate in Happy Cell ASM was lower than that in Matrigel and gradually decreased as passage numbers increased (data not shown); therefore, the current suspension culture would be suitable for culture expansion immediately before performing large-scale assays. Notably, despite the probable higher proportion of differentiated IECs and lower proportion of IESCs, suspension-cultured organoids can respond to IL-22 and WNT3A, which indicates that drug screening, focusing on intestinal epithelial cell function and growth of IESCs, can be properly performed, and these cytokines can be used to monitor or control the assay quality. Moreover, DNA microarray analysis revealed that the expression of many other genes can fluctuate following the suspension culture ([Tables S1](#mmc2){ref-type="supplementary-material"} and [S2](#mmc3){ref-type="supplementary-material"}). The significance of these changes is so far unclear; however, it may be a clue to resolve the issue for the improvement of organoid suspension culture. Meanwhile, we observed that WNT3A supplementation enhanced expression of stem cell markers (*LGR5* and *ASCL2*), and concomitantly increased MKI67^+^ proliferative cells in whole-mount organoids. Nevertheless, the number of MKI67^+^ cells ([Figure 6](#fig6){ref-type="fig"}C) is still smaller than that cultured in Matrigel ([Figure 2](#fig2){ref-type="fig"}I); therefore, further modification of suspension culture medium components, such as supplementation of suitable ECM and small molecules/compounds, will be needed to improve IESC growth. In addition, it is of interest to investigate other 3D culture technologies, such as use of scaffold-based culture plates and bioreactors, to further improve conditions for organoid culture ([@bib16]).

In summary, we have developed a systematic methodology for human iPSC-derived intestinal organoid culture that is more convenient and cost-effective than currently available methods. The methods introduced in this study will aid in promoting organoid studies by permitting applications requiring large numbers of organoids, such as gene and therapeutic drug screening and translational research.

Experimental Procedures {#sec4}
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Materials {#sec4.1}
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All materials used in this study are listed in [Table S3](#mmc1){ref-type="supplementary-material"}.

Mice {#sec4.2}
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C57BL/6J mice were purchased from CLEA Japan. The institutional animal care and research advisory committee at the University of Tokyo approved all animal procedures.

Cell Culture and Differentiation Experiments {#sec4.3}
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All cell culture and differentiation procedures of human iPSCs into intestinal organoids are described in the [Supplemental Experimental Procedures](#mmc1){ref-type="supplementary-material"}.

Preparation of Human Intestinal Organoids {#sec4.4}
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Human intestinal crypts were isolated as described previously ([@bib33]), with minor modifications. The healthy intestinal tissues (ileum and colon) of surgical specimens were chopped into approximately 5-mm pieces and washed with cold PBS. The fragmented tissues were then incubated in cold chelation buffer ([Table S4](#mmc1){ref-type="supplementary-material"}) for 30 min on ice. Following removal of the chelation buffer, the tissue fragments were resuspended with cold PBS and vigorously mixed in a 50-mL tube for 40 s, and the supernatant was then centrifuged at 200 × *g* for 3 min. This resuspension/centrifugation procedure was usually performed eight times. The pellets were suspended with PBS, passed through 150-μm nylon filters, centrifuged at 300 × *g* for 3 min, and further passed through 100-μm nylon filters. Crypt culture largely followed the previously described protocol of the Tokyo Medical and Dental University ([@bib45]), except that it was embedded in Matrigel rather than type I collagen gels. The experiments were approved by the human ethical committee of The University of Tokyo and Osaka University, and all tissues were sampled with informed consent.

Organoid Culture and Passage {#sec4.5}
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Organoid culture and passage in Matrigel were performed as described previously ([@bib38]). When using Happy Cell ASM (Biocroi, Ireland), disrupted organoids were suspended with Happy Cell human organoid culture medium ([Table S4](#mmc1){ref-type="supplementary-material"}) and seeded on ultra-low-attachment cell culture plate (Corning, 3473) (0.5 mL/well). Equal amounts of medium were also supplemented every 3 days, and organoids were collected every 6 days by centrifugation at 440 × *g* for 5 min after adding 4 mL of Dulbecco\'s modified Eagle\'s medium (DMEM). For the IL-22 stimulation assays, the culture medium was replaced with pre-stimulation medium or Happy Cell pre-stimulation medium ([Table S4](#mmc1){ref-type="supplementary-material"}) containing human IL-22 with various concentrations for 2 days.

Lentiviral Infection {#sec4.6}
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Lentiviral expression plasmids for mWNT3A, hRSPO1, hNOG, human HGF, and human IL-22 were constructed by inserting the full-length PCR fragment into CSII-EF-MCS-IRES2-Venus, which is provided by Dr. Hiroyuki Miyoshi (BioResource Center, RIKEN, Japan). Following detail procedures are described in the [Supplemental Experimental Procedures](#mmc1){ref-type="supplementary-material"}.

Organoid Regeneration from 2D-Cultured Cells {#sec4.7}
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After harvesting organoids from Matrigel, they were disrupted using a 29G needle 10 times, seeded in collagen I-coated 12-well plates, and cultured with human organoid culture medium to obtain proliferative cells. Proliferative cells (including IESCs) were then infected with lentiviruses or transfected with Lipofectamine 2000 (Thermo Fisher Scientific), trypsinized for 5 min, and harvested. After centrifugation at 440 × *g* for 5 min, they were resuspended in Matrigel with 20% human organoid culture medium on ice, and suspensions were aliquoted into the wells of a 24-well plate. They were cultured as normal organoid passages.

Statistical Analysis {#sec4.8}
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All results are presented as mean ± SEM. Data were analyzed using Student\'s t test for two groups and Dunnett\'s test for ≥3 groups to compare all groups with a control group. Differences were considered significant at p \< 0.05.

Author Contributions {#sec5}
====================

Y.T., S.S., T. Yamamoto, and S.K. performed the experiments. Y.K., Y.Y., T. Yamaguchi, and M.H. supported the data analysis and provided helpful discussions. N.T., S.U., C.-Y.L., M.O., H.M., H.O., T.M., and J.N. managed and provided human samples including iPSCs and surgical specimens. Y.T., S.S., and H.K. designed and directed the study and wrote the paper. All authors approved the final version of the manuscript.

Supplemental Information {#app2}
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Document S1. Supplemental Experimental Procedures, Figures S1--S4, and Tables S3 and S4Table S1. Representative Upregulated Genes in TkDN4-M Organoids When Cultured in Happy Cell ASM Compared with in Matrigel, Related to Figure 6Table S2. Representative Downregulated Genes in TkDN4-M Organoids When Cultured in Happy Cell ASM Compared with in Matrigel, Related to Figure 6Document S2. Article plus Supplemental Information

We thank Dr. Atsushi Miyawaki (Brain Science Institute, RIKEN, Japan) for providing us Venus-containing plasmids and Drs. Haruna Takagi, Chizuru Tsuzuki, and Ayae Nishiyama for technical support. The project is supported by a joint grant from Japan Tobacco and grants from the Ministry of Education, Science, Sports and Technology of Japan (Grant-in Aid for Young Scientists B \[25860353 to S.S.\] and for Scientific Research S \[23229004 to H.K.\]) and the Japan Agency for Medical Research and Development (AMED) (to H.K.).

Supplemental Information includes Supplemental Experimental Procedures, four figures, and four tables and can be found with this article online at [https://doi.org/10.1016/j.stemcr.2017.11.004](10.1016/j.stemcr.2017.11.004){#intref0010}.

[^1]: Co-senior author

[^2]: Present address: Mucosal Vaccine Project, BIKEN Innovative Vaccine Research Alliance Laboratories, Research Institute for Microbial Diseases, Osaka University, Osaka 565--0871, Japan