The Intestinal Stem Cell Niche: Generation and Utilization of Intestinal Organoids

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The literature review by Takahashi and Takase describes the cellular components of the intestinal stem cell niche – both mesenchymal and epithelial – and the various signaling factors that they secrete to support maintenance of intestinal stem cells. The authors also discuss current methods for murine 3D organoid cultures and 2D organoid monolayers. Overall, the manuscript is not very cohesive. While some sections are detailed and informative, other sections are too brief.

 

Major comments: 

1. Section 2 (ISC niche) and section 3 (organoids) are rather disjointed. The detailed description of different cell types and subtypes in section 2 is informative, however, section 3 is quite vague. Also, there is a lot of emphasis on Wnt ligands in the mesenchymal niche subsection, but exogenous Wnt is actually dispensable for murine small intestinal organoid culture. Section 3 should be expanded, and it needs to better relate back to the information presented in Section 2.

2. The description of 2D organoid monolayers is also superficial. There were multiple studies on murine 2D organoid monolayers published prior to the authors’ previous publication, and these should be described and cited. Any differences between established methods could be discussed.

3. The second paragraph in subsection 3.2 does not tie into this section and should be revised.

4. The last two-thirds of the Conclusions paragraph also does not relate well to the rest of the manuscript and should be rewritten. The authors could consider including a section on Future Directions.

 

Minor comments: 

1. Please clarify the discussion regarding BMP signaling and PDGFalpha+ cells because BMP ligands are thought to promote ISC differentiation rather than maintain stemness.

2. The section heading 3 “Organogenesis in the small intestine” does not accurately describe the section.

3. Subsection title 3.1 “D intestinal organoids” (typo)

4. Review papers are not supposed to include new unpublished data, as appear in Figure 3B and C. If these data are reproduced from a previous publication, then that should be indicated and cited appropriately.

Author Response

Comments and Suggestions for Authors

The literature review by Takahashi and Takase describes the cellular components of the intestinal stem cell niche – both mesenchymal and epithelial – and the various signaling factors that they secrete to support maintenance of intestinal stem cells. The authors also discuss current methods for murine 3D organoid cultures and 2D organoid monolayers. Overall, the manuscript is not very cohesive. While some sections are detailed and informative, other sections are too brief.

Response: Thank you for your constructive comments.

 

Major comments: 

1. Section 2 (ISC niche) and section 3 (organoids) are rather disjointed. The detailed description of different cell types and subtypes in section 2 is informative, however, section 3 is quite vague. Also, there is a lot of emphasis on Wnt ligands in the mesenchymal niche subsection, but exogenous Wnt is actually dispensable for murine small intestinal organoid culture. Section 3 should be expanded, and it needs to better relate back to the information presented in Section 2.

Response: According to the reviewer’s comment, we added information in Section 3 to better relate back to the information presented in Section 2 as follows.

“The intestinal stem niche is like a cradle of ISCs for maintaining and controlling the activity in gut. The specialized functions required to ensure proper ISC functions are provided by neighboring differentiated cells including Paneth and mesenchymal cells.　Through signaling pathways and intercellular interactions, these cells influence the behavior of adjacent ISCs, which themselves exhibit relatively limited specialization. Consequently, ISC identity is defined more by spatial localization within the niche rather than by distinct gene expression patterns. To characterize ISCs within their niche, researchers analyze the surrounding cellular environment, including neighboring cell types, signaling molecule expression, and extracellular matrix components. Once commonalities are found, the niche can be perturbed to learn which aspects affect ISC behavior. More understanding niches and their signals will provide valuable insights into why most of the characterized niche regulatory mechanisms and signals also function in developing embryos. Unlike embryonic cells, ISCs can operate in a steady state. They can generate on average one replacement ISC and one tissue cell at each division with no apparent limit. For over a decade, intestinal organoids have been utilized to mimic villus and crypt development in vitro [18]. Most research has been developed culturing organoids in three-dimensional organoids that form crypt-like structures that grow stochastically and uncontrollably. When cultured on two-dimensional flat elastic substrates, organoids only form flat crypt-like structures [81]. In this section, we will describe generation and recent utilization of intestinal organoids.”

 

Additionally, we added new sentences concerning the role of Wnt3s in intestinal organoid culture as follows.

“Murine intestinal organoids, unlike their human, can be cultured without the addition of exogenous Wnt3a [18,72]. However, intestinal organoids derived from Wntless-deficient mice, which lack the ability to secrete Wnt ligands extracellularly, cannot proliferate in the absence of Wnt3a supplementation [82]. This finding suggests that in mice, Wnt ligands secreted by IECs, primarily Paneth cells, are sufficient to sustain organoid growth by providing the necessary Wnt signaling.”

We added references as follows.

[81] Pérez-González, C.; Ceada1, G.; Greco, F.; Matejčić1, M.; Gómez-González, M.; Castro1, N.; Menendez, A.; Kale, S.; Krndija, D.; Clark, A.G.; Mechanical compartmentalization of the intestinal organoid enables crypt folding and collective cell migration. Nat. Cell Biol. 2021, 23, 745–757.

[82] Gao, G.; Wei, G.; Liu, S.; Chen, J.; Zeng, Z.; Zhang, X.; Chen, F.; Zhuo, L.; Hsu, W.; Li, D.; Liu, M.; & Zhang, X. Epithelial Wntless is dispensable for intestinal tumorigenesis in mouse models. Biochem. Biophys. Res. Commun. 2019, 519, 754–760.

 

We also expanded Section3.2. as follows.

‘The development of the 3D intestinal organoid culture method has significantly advanced our understanding of ISCs and their niche environment. However, it should be noted that 3D organoids are not a universal experimental model for intestinal research. One major limitation is the orientation of epithelial polarity. In 3D organoids, the apical surface of epithelial cells, which interacts with luminal factors such as dietary components and microbes, faces inward toward the organoid lumen, rendering direct access to this interface experimentally challenging. Additionally, these structures lack distinct villus formations, which are critical for modeling intestinal physiology. To overcome these constraints, various experimental approaches have been explored. For example, methods of injecting substances into the lumen of a 3D organoid or turning the organoid inside out have been reported [90,91]. However, these methods are difficult to perform and inefficient. Additionally, monolayer cultures derived from human colorectal cancer cell lines (Caco-2) have been utilized as analytical systems [92,93]. However, Caco-2 monolayers have been reported to exhibit gene expression and physiological characteristics distinct from those of the native small intestinal epithelium [93,94]. Given these challenges, recent research has focused on developing 2D epithelial monolayers derived from 3D intestinal organoids as an alternative experimental system to overcome these limitations.

The generation of 2D small intestinal epithelial monolayers has been attempted in multiple species, including humans, mice, pigs, and bovines [94-100]. In particular, for human-derived monolayers, several research groups have reported methods using 3D intestinal organoids or iPSC-derived IECs [94-96]. In contrast, generating 2D monolayers from mouse 3D intestinal organoids has been challenging due to the rapid turnover of IECs. In a few cases, methods requiring primary tissue (myofibroblast or enteric nervous system)-derived culture medium or long culture periods have been reported [99,10097,98]. Recently, we developed a method for efficiently forming 2D epithelial monolayers from murine 3D small intestinal organoids using only commercially available reagents and media [101] (Figure 3A). The 2D epithelial monolayers have stable intercellular junctions and contain ISCs and mature IECs [101] (Figure 3B and C). Moreover, the transepithelial electric resistance values of the monolayer were within the expected physiological range [101,102]. This new method is expected to be useful in physiology and pharmaceutical research.

Additionally, 2D intestinal epithelial monolayers, when combined with various experimental approaches, have the potential to provide novel insights into the mechanisms underlying intestinal epithelial morphogenesis. During development, the intestinal epithelium initially forms a flat structure, which subsequently gives rise to characteristic villus and crypt structures through interactions with surrounding tissues [1,103,104]. Recent studies have reported that human 2D intestinal monolayers, when cultured under ALI conditions or subjected to shear forces via shaking culture, which mimics intestinal luminal flow, can induce the formation of villus-like protrusions [105,106]. The intestinal epithelium with its specialized cell organization is intricately folded into arrays of crypts and villi. While the mechanisms driving villi formation are well described, the processes regulating crypt formation remain largely unknown. Pérez-González and coworkers found that the size of ISC compartment depends on the extracellular-matrix stiffness and endogenous cellular forces [81]. Additionally, computational modelling revealed that crypt shape and force distribution rely on cell surface tensions following cortical actomyosin density. They will clarify crypt morphogenesis using 2D intestinal monolayers in the near future. Eicher and colleagues successfully established a co-culture system by separately differentiating endoderm and mesoderm from human iPS cells to generate gastrointestinal tissue-like structures [107]. More recently, Deguchi and colleagues developed a micro-small intestine system by simultaneously differentiating definitive endoderm and mesoderm from human iPS cells on a microfluidic device that replicates intestinal flow. This system enabled the formation of a 3D small-intestine-like tissue with villus-like epithelium and an aligned mesenchymal layer [108]. However, it has not yet fully recapitulated the mature crypt structure. Analysis systems employing microfluidic devices and stiffness-controlled artificial hydrogels to constrain the shape of 2D monolayers and 3D organoids have provided valuable insights into the mechanical properties of ISCs and their niche during intestinal morphogenesis [109-111]. While none of these experimental models perfectly recapitulate the in vivo situation, they continue to advance our understanding of various aspects of intestinal biology.”

 

We added new references as follows.

[81] Pérez-González, C.; Ceada1, G.; Greco, F.; Matejčić1, M.; Gómez-González, M.; Castro1, N.; Menendez, A.; Kale, S.; Krndija, D.; Clark, A.G.; Mechanical compartmentalization of the intestinal organoid enables crypt folding and collective cell migration. Nat. Cell Biol. 2021, 23, 745–757.

[90] Wilson, S.S.; Tocchi, A.; Holly, M.K.; Parks, W.C.; Smith, J.G.A. Small Intestinal Organoid Model of Non-Invasive Enteric Pathogen-Epithelial Cell Interactions. Mucosal Immunol 2015, 8, 352–361.

[91] Co, J.Y.; Margalef-Català, M.; Li, X.; Mah, A.T.; Kuo, C.J.; Monack, D.M.; Amieva, M.R. Controlling Epithelial Polarity: A Human Enteroid Model for Host-Pathogen Interactions. Cell Rep 2019, 26, 2509-2520.e4.

[92] Sambuy, Y.; De Angelis, I.; Ranaldi, G.; Scarino, M.L.; Stammati, A.; Zucco, F. The Caco-2 Cell Line as a Model of the Intestinal Barrier: Influence of Cell and Culture-Related Factors on Caco-2 Cell Functional Characteristics. Cell Biology and Toxicology. 2005, 21, 1–26.

[93] Sun, H.; Chow, E.C.Y.; Liu, S.; Du, Y.; Pang, K.S. The Caco-2 Cell Monolayer: Usefulness and Limitations. Expert Opinion on Drug Metabolism and Toxicology. 2008, 4, 395–411.

[94] Negoro, R.; Takayama, K.; Kawai, K.; Harada, K.; Sakurai, F.; Hirata, K.; Mizuguchi, H. Efficient Generation of Small Intestinal Epithelial-like Cells from Human IPSCs for Drug Absorption and Metabolism Studies. Stem Cell Reports 2018, 11, 1539–1550.

[95] Sasaki, N.; Miyamoto, K.; Maslowski, K.M.; Ohno, H.; Kanai, T.; Sato, T. Development of a Scalable Coculture System for Gut Anaerobes and Human Colon Epithelium. Gastroenterology 2020, 159, 388-390.e5.

[96] Takahashi, Y.; Noguchi, M.; Inoue, Y.; Sato, S.; Shimizu, M.; Kojima, H.; Okabe, T.; Kiyono, H.; Yamauchi, Y.; Sato, R. Organoid-Derived Intestinal Epithelial Cells Are a Suitable Model for Preclinical Toxicology and Pharmacokinetic Studies. iScience 2022, 25, 104542.

[97] Puzan, M.; Hosic, S.; Ghio, C.; Koppes, A. Enteric Nervous System Regulation of Intestinal Stem Cell Differentiation and Epithelial Monolayer Function. Sci Rep 2018, 8, 6313.

[98] Altay, G.; Larrañaga, E.; Tosi, S.; Barriga, F.M.; Batlle, E.; Fernández-Majada, V.; Martínez, E. Self-Organized Intestinal Epithelial Monolayers in Crypt and Villus-like Domains Show Effective Barrier Function. Sci Rep 2019, 9, 10140.

[99] Hoffmann, P.; Schnepel, N.; Langeheine, M.; Kunnemann, K.; Grassl, G.A.; Brehm, R.; Seeger, B.; Mazzuoli-Weber, G.; Breves, G. Intestinal Organoid-Based 2D Monolayers Mimic Physiological and Pathophysiological Properties of the Pig Intestine. PLoS One 2021, 16, e0256143.

[100] Kawasaki, M.; Ambrosini, Y.M. Accessible Luminal Interface of Bovine Rectal Organoids Generated from Cryopreserved Biopsy Tissues. PLoS One 2024, 19, e0301079.

[104] Felsenthal, N.; Vignjevic, D.M. Stand by Me: Fibroblasts Regulation of the Intestinal Epithelium during Development and Homeostasis. Current Opinion in Cell Biology. 2022, 78, 102116.

[105] Onozato, D.; Ogawa, I.; Kida, Y.; Mizuno, S.; Hashita, T.; Iwao, T.; Matsunaga, T. Generation of Budding-Like Intestinal Organoids from Human Induced Pluripotent Stem Cells. J Pharm Sci 2021, 110, 2637-2650.

[106] Sugimoto, S.; Kobayashi, E.; Fujii, M.; Ohta, Y.; Arai, K.; Matano, M.; Ishikawa, K.; Miyamoto, K.; Toshimitsu, K.; Takahashi, S.; et al. An Organoid-Based Organ-Repurposing Approach to Treat Short Bowel Syndrome. Nature 2021, 592, 99-104.

[109] Pentinmikko, N.; Lozano, R.; Scharaw, S.; Andersson, S.; Englund, J.I.; Castillo-Azofeifa, D.; Gallagher, A.; Broberg, M.; Song, K.Y.; Carvajal, A.S.; et al. Cellular Shape Reinforces Niche to Stem Cell Signaling in the Small Intestine. Sci Adv 2022, 8, eabm1847.

[110] Yavitt, F.M.; Kirkpatrick, B.E.; Blatchley, M.R.; Speckl, K.F.; Mohagheghian, E.; Moldovan, R.; Wang, N.; Dempsey, P.J.; Anseth, K.S. In Situ Modulation of Intestinal Organoid Epithelial Curvature through Photoinduced Viscoelasticity Directs Crypt Morphogenesis. Sci Adv 2023, 9, eadd5668.

[111] Baghdadi, M.B.; Houtekamer, R.M.; Perrin, L.; Rao-Bhatia, A.; Whelen, M.; Decker, L.; Bergert, M.; Pérez-Gonzàlez, C.; Bouras, R.; Gropplero, G.; et al. PIEZO-Dependent Mechanosensing Is Essential for Intestinal Stem Cell Fate Decision and Maintenance. Science 2024, 386, eadj7615.

 

2. The description of 2D organoid monolayers is also superficial. There were multiple studies on murine 2D organoid monolayers published prior to the authors’ previous publication, and these should be described and cited. Any differences between established methods could be discussed.

Response: Thank you for your constructive comment. Accordingly, we rewritten Subsection 3.2 profoundly. Please see our response to Comment 1.

 

3. The second paragraph in subsection 3.2 does not tie into this section and should be revised.

Response: Accordingly, we revised the second paragraph in subsection 3.2. Please see our response to Comment 1.

 

4. The last two-thirds of the Conclusions paragraph also does not relate well to the rest of the manuscript and should be rewritten. The authors could consider including a section on Future Directions.

Response: Thank you for your constructive comment. Accordingly, we added new sentences, and a new paragraph related to the rest of our manuscript and changed Section 4 title to “Conclusions and future directions” as follows.

“For instance, the technique for conversion from colon organoids into small intestinal tissues is transformative for the development of tissue engineering applications for diseases such as short bowel syndrome [106]. Additionally, many intestinal diseases show changes in crypt morphology including inflammatory bowel disease and irritable bowel disease. These diseases represent an important health burden affecting a significant percentage of the population. The understanding of the mechanisms that regulate crypt malformations in the intestinal stem cell niche may provide critical information to progress in disease treatment and drug development.

                 We discussed ISC-derived organoid culture including their methodology, application, and weakness of intestinal organoids and engineered organoids compared to cell lines. The appropriate applications, advantages and disadvantages of the model have been articulated in Section 3. The development of 2D epithelial monolayers as well as intestinal 3D organoids provides an unprecedented tool for humans to study diseases. By combining microfluidic technology, innovative biological support materials such as extracellular matrices, automated detection methods using AI technologies, we have a more faithful promise that intestinal organoid technology will greatly accelerate the drug discovery process for intestinal diseases and the innovation of treatment methods”

 

Minor comments: 

1. Please clarify the discussion regarding BMP signaling and PDGFalpha+ cells because BMP ligands are thought to promote ISC differentiation rather than maintain stemness.

Response: Accordingly, we added new sentences and a new reference as follows.

“A complex gradient of BMP signaling formed by PDGFα+ cells promotes ISC differentiation rather than maintains ISC stemness. BMP signaling is low in the crypt base by PDGFαlow cells and higher towards the top of the villi by PDGFαhigh cells [47]. The gradient inhibits proliferation of the Lgr5+ ISCs via Smad-mediated repression of genes including Lgr5. Eventually, BMP signaling promotes ISC differentiation [47].”

[47] Kraiczy, J.; McCarthy, N.; Malagola, E.; Tie, G.; Madha, S.; Boffelli, D.; Wagner, D.E.; Wang, T.C.; Shivdasani, R.A. Graded BMP signaling within intestinal crypt architecture directs self-organization of the Wnt-secreting stem cell niche. Cell Stem Cell 2023, 30, 433-449.

 

2. The section heading 3 “Organogenesis in the small intestine” does not accurately describe the section.

Response: According to the reviewer’s comment, we added information in Section 3. Please see our response to Comment 1.

 

3. Subsection title 3.1 “D intestinal organoids” (typo)

Response: We corrected as “Three-dimensional (3D) intestinal organoids.”

 

4. Review papers are not supposed to include new unpublished data, as appear in Figure 3B and C. If these data are reproduced from a previous publication, then that should be indicated and cited appropriately.

Response: According to the reviewer’s comment, we cited previous publications for reproducing the data of Figure 3B and C as follows.

(Figure 3): “Fluorescent images have been compiled from our published data [101].”

[101] Takase, Y.; Takahashi, T. Method for two-dimensional epithelial monolayer formation derived from mouse three-dimensional small intestinal organoids. Methods Mol. Biol. 2024, 2749, 73-84.

Reviewer 2 Report

Comments and Suggestions for Authors

In this review, the authors provide an overview of intestinal stem cells and their associated niches, including both mesenchymal and epithelial niches. Additionally, they discuss organoids derived from intestinal stem cells. However, the review does not incorporate the most recent research advancements related to intestinal stem cells and organoid culture systems. To improve the comprehensiveness and relevance of this review, it is recommended that the authors include more up-to-date studies published in the field, particularly in high-impact journals.

Comments:

1.          The authors should include a broader introduction to the diversity of intestinal stem cells identified in prior research. While Lgr5+ stem cells represent a pivotal and well-studied population, they are only one type among several intestinal stem cell populations. Additional focus on other stem cell types would enhance the comprehensiveness of the review.

2.          A detailed summary of the differences among various mesenchymal populations in the intestinal niche is needed. This should include their overlapping roles and expression of key signals that are critical for supporting intestinal stem cell niches. A diagram illustrating these relationships and signaling pathways would make this section more accessible and clear.

 

3.          The section on stem cell-derived organoid culture systems would benefit from expansion. The authors should discuss these systems in more detail, including their methodology, applications, and the research questions they aim to address. Including the most recent advances and publications in this field, particularly those from high-impact journals, would significantly improve the quality and relevance of the review.

Author Response

Comments and Suggestions for Authors

In this review, the authors provide an overview of intestinal stem cells and their associated niches, including both mesenchymal and epithelial niches. Additionally, they discuss organoids derived from intestinal stem cells. However, the review does not incorporate the most recent research advancements related to intestinal stem cells and organoid culture systems. To improve the comprehensiveness and relevance of this review, it is recommended that the authors include more up-to-date studies published in the field, particularly in high-impact journals.

Response: Thank you for your constructive comments.

 

Comments:

1. The authors should include a broader introduction to the diversity of intestinal stem cells identified in prior research. While Lgr5+ stem cells represent a pivotal and well-studied population, they are only one type among several intestinal stem cell populations. Additional focus on other stem cell types would enhance the comprehensiveness of the review.

Response: According to the reviewer’s comment, we added new sentences and references to describe a broader introduction to the diversity of ISCs identified in prior research as follows.

"In addition to Lgr5+ ISCs, the existence of functionally distinct populations of intestinal stem cells has long been a hotly debated topic with many research groups dedicating themselves to this question [10-17]. The highly proliferative Lgr5+ ISCs can also be divided into sub-populations [11].”

[10] Ayyaz, A,; Kumar, S.; Sangiorgi, B.; Ghoshal, B.; Gosio, J.; Ouladan, S,; Fink, M.; Barutcu, S.; Trcka, D.; Shen, J.; et al. Single-cell transcriptomes of the regenerating intestine reveal a revival stem cell. Nature 2019, 569, 121-125.

[11] Barriga, F.M.; Montagni, E.; Mana, M.; Guillaumet-Adkins, A.; Hernando-Momblona, X.; Sevillano, M.; Rodriguez-Esteban, G.; Mendez-Lago, M.; Buczacki, S.J.A.; Gut, Ivo.; et al. Mex3a marks slow-proliferating multilineage progenitors of the intestinal epithelium. Cell Stem Cell 2017, 20, 801-816.e7.

[12] Li, N.; Yousefi, M.; Nakauka-Ddamba, A.; Jain, R.; Tobias, J.; Epstein, J.A.; Jensen, S.T.; Lengner, C.J. Single-Cell Analysis of Proxy Repor ter Allele-Marked Epithelial Cells Establishes Intestinal Stem Cell Hierarchy. Stem Cell Rep. 2014, 3, 876-891.

[13] May, R.; Sureban, S.M.; Hoang, N.; Riehl, T.E.; Lightfoot, S.A.; Ramanujam, R.; Wyche, J. H.; Anant, S.; Houchen, C.W. Doublecortin and CaM kinase-like-1 and leucine-rich-repeat-containing G-protein-coupled receptor mark quiescent and cycling intestinal stem cells, respectively. Stem Cells Dayt Ohio 27, 2009, 2571–2579.

[14] Metcalfe, C.; Kljavin, N.M.; Ybarra, R.; de Sauvage, F.J. Lgr5+ stem cells are indispensable for radiation-induced intestinal regeneration. Cell Stem Cell 2014, 14, 149-159.

[15] Montgomery, R.K.; Carlone, D.L.; Richmond, C.A.; Farilla, L.; Kranendonk, M.E.G.; Henderson, D.E.; Baffour-Awuah, N.Y.; Ambruzs, D.M.; Fogli, L.K.; Algra, S.; et al. Mouse telomerase reverse transcriptase (mTert) expression marks slowly cycling intestinal stem cells. Proc. Natl. Acad. Sci. USA 2011,108, 179–184.

[16] Roche, K.C.; Gracz, A.D.; Liu, X.F.; Newton, V.; Akiyama, H.; Magness, S.T. SOX9 maintains reserve stem cells and preserves radioresistance in mouse small intestine. Gastroenterology 2015, 149, 1553–1563.e10.

[17] Yan, K.S.; Chia, L.A.; Li, X.; Ootani, A.; Su, J.; Lee, J.Y.; Su, N.; Luo, Y.; Heilshorn, S.C.; Amieva, M.R.; The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations. Proc. Natl. Acad. Sci. USA 2012, 109, 466-471.

 

2. A detailed summary of the differences among various mesenchymal populations in the intestinal niche is needed. This should include their overlapping roles and expression of key signals that are critical for supporting intestinal stem cell niches. A diagram illustrating these relationships and signaling pathways would make this section more accessible and clear.

Response: According to the reviewer’s comment, we added signaling pathways in Figure 2.

 

3. The section on stem cell-derived organoid culture systems would benefit from expansion. The authors should discuss these systems in more detail, including their methodology, applications, and the research questions they aim to address. Including the most recent advances and publications in this field, particularly those from high-impact journals, would significantly improve the quality and relevance of the review.

Response: Thank you for your constructive comment. Accordingly, we expanded discussion of “Organogenesis in the small intestine” as follows.

• Three-dimensional (3D) intestinal organoids

We added a new paragraph and references as follows.

“As the emergence of cutting-edge technologies including 3D bioprinting or microfluidic chips raises the prospect of closely mimicking the microenvironment [87], current research frequently focuses on combinations of IECs or mesenchymal cells. Multi-organ chips containing vascularization and immunization that connect multiple organ components appear to be better models for studying drug transport and metabolism [88,89].”

 (References)

[87] Radhakrishnan, J.; Varadaraj, S.; Dash, S.K.; Sharma, A.; Verma, R.S. Organotypic

cancer tissue models for drug screening: 3D constructs, bioprinting and microfluidic chips. Drug Discov. Today 2020, 25, 879–890.

[88] Shirure, V.S.; Hughes, C.C.W.; George, S.C. Engineering vascularized organoid-on-a-chip models. Annu. Rev. Biomed. Eng. 2021, 23, 141–167.

[89] Schafer, S.T.; Mansour, A.A.; Schlachetzki, J.C.M.; Pena, M.; Ghassemzadeh, S.; Mitchell, L.; Mar, A.; Quang, D.; Stumpf, S.; Ortiz, I.S.; et al. An in vivo neuroimmune organoid model to study human microglia phenotypes. Cell 2023, 186, 2111-2126.e2120.

 

• Two-dimensional (2D) epithelial monolayers

We rewritten Subsection 3.2 profoundly as follows.

“The development of the 3D intestinal organoid culture method has significantly advanced our understanding of ISCs and their niche environment. However, it should be noted that 3D organoids are not a universal experimental model for intestinal research. One major limitation is the orientation of epithelial polarity. In 3D organoids, the apical surface of epithelial cells, which interacts with luminal factors such as dietary components and microbes, faces inward toward the organoid lumen, rendering direct access to this interface experimentally challenging. Additionally, these structures lack distinct villus formations, which are critical for modeling intestinal physiology. To overcome these constraints, various experimental approaches have been explored. For example, methods of injecting substances into the lumen of a 3D organoid or turning the organoid inside out have been reported [90,91]. However, these methods are difficult to perform and inefficient. Additionally, monolayer cultures derived from human colorectal cancer cell lines (Caco-2) have been utilized as analytical systems [92,93]. However, Caco-2 monolayers have been reported to exhibit gene expression and physiological characteristics distinct from those of the native small intestinal epithelium [93,94]. Given these challenges, recent research has focused on developing 2D epithelial monolayers derived from 3D intestinal organoids as an alternative experimental system to overcome these limitations.

The generation of 2D small intestinal epithelial monolayers has been attempted in multiple species, including humans, mice, pigs, and bovines [94-100]. In particular, for human-derived monolayers, several research groups have reported methods using 3D intestinal organoids or iPSC-derived IECs [94-96]. In contrast, generating 2D monolayers from mouse 3D intestinal organoids has been challenging due to the rapid turnover of IECs. In a few cases, methods requiring primary tissue (myofibroblast or enteric nervous system)-derived culture medium or long culture periods have been reported [97,98]. Recently, we developed a method for efficiently forming 2D epithelial monolayers from murine 3D small intestinal organoids using only commercially available reagents and media [101] (Figure 3A). The 2D epithelial monolayers have stable intercellular junctions and contain ISCs and mature IECs [101] (Figure 3B and C). Moreover, the transepithelial electric resistance values of the monolayer were within the expected physiological range [101,102]. This new method is expected to be useful in physiology and pharmaceutical research.

Additionally, 2D intestinal epithelial monolayers, when combined with various experimental approaches, have the potential to provide novel insights into the mechanisms underlying intestinal epithelial morphogenesis. During development, the intestinal epithelium initially forms a flat structure, which subsequently gives rise to characteristic villus and crypt structures through interactions with surrounding tissues [1,103,104]. Recent studies have reported that human 2D intestinal monolayers, when cultured under ALI conditions or subjected to shear forces via shaking culture, which mimics intestinal luminal flow, can induce the formation of villus-like protrusions [105,106]. The intestinal epithelium with its specialized cell organization is intricately folded into arrays of crypts and villi. While the mechanisms driving villi formation are well described, the processes regulating crypt formation remain largely unknown. Pérez-González and coworkers found that the size of ISC compartment depends on the extracellular-matrix stiffness and endogenous cellular forces [81]. Additionally, computational modelling revealed that crypt shape and force distribution rely on cell surface tensions following cortical actomyosin density. They will clarify crypt morphogenesis using 2D intestinal monolayers in the near future. Eicher and colleagues successfully established a co-culture system by separately differentiating endoderm and mesoderm from human iPS cells to generate gastrointestinal tissue-like structures [107]. More recently, Deguchi and colleagues developed a micro-small intestine system by simultaneously differentiating definitive endoderm and mesoderm from human iPS cells on a microfluidic device that replicates intestinal flow. This system enabled the formation of a 3D small-intestine-like tissue with villus-like epithelium and an aligned mesenchymal layer [108]. However, it has not yet fully recapitulated the mature crypt structure. Analysis systems employing microfluidic devices and stiffness-controlled artificial hydrogels to constrain the shape of 2D monolayers and 3D organoids have provided valuable insights into the mechanical properties of ISCs and their niche during intestinal morphogenesis [109-111]. While none of these experimental models perfectly recapitulate the in vivo situation, they continue to advance our understanding of various aspects of intestinal biology.

’

We added new references as follows.

[81] Pérez-González, C.; Ceada1, G.; Greco, F.; Matejčić1, M.; Gómez-González, M.; Castro1, N.; Menendez, A.; Kale, S.; Krndija, D.; Clark, A.G.; Mechanical compartmentalization of the intestinal organoid enables crypt folding and collective cell migration. Nat. Cell Biol. 2021, 23, 745–757.

[90] Wilson, S.S.; Tocchi, A.; Holly, M.K.; Parks, W.C.; Smith, J.G.A. Small Intestinal Organoid Model of Non-Invasive Enteric Pathogen-Epithelial Cell Interactions. Mucosal Immunol 2015, 8, 352–361.

[91] Co, J.Y.; Margalef-Català, M.; Li, X.; Mah, A.T.; Kuo, C.J.; Monack, D.M.; Amieva, M.R. Controlling Epithelial Polarity: A Human Enteroid Model for Host-Pathogen Interactions. Cell Rep 2019, 26, 2509-2520.e4.

[92] Sambuy, Y.; De Angelis, I.; Ranaldi, G.; Scarino, M.L.; Stammati, A.; Zucco, F. The Caco-2 Cell Line as a Model of the Intestinal Barrier: Influence of Cell and Culture-Related Factors on Caco-2 Cell Functional Characteristics. Cell Biology and Toxicology. 2005, 21, 1–26.

[93] Sun, H.; Chow, E.C.Y.; Liu, S.; Du, Y.; Pang, K.S. The Caco-2 Cell Monolayer: Usefulness and Limitations. Expert Opinion on Drug Metabolism and Toxicology. 2008, 4, 395–411.

[94] Negoro, R.; Takayama, K.; Kawai, K.; Harada, K.; Sakurai, F.; Hirata, K.; Mizuguchi, H. Efficient Generation of Small Intestinal Epithelial-like Cells from Human IPSCs for Drug Absorption and Metabolism Studies. Stem Cell Reports 2018, 11, 1539–1550.

[95] Sasaki, N.; Miyamoto, K.; Maslowski, K.M.; Ohno, H.; Kanai, T.; Sato, T. Development of a Scalable Coculture System for Gut Anaerobes and Human Colon Epithelium. Gastroenterology 2020, 159, 388-390.e5.

[96] Takahashi, Y.; Noguchi, M.; Inoue, Y.; Sato, S.; Shimizu, M.; Kojima, H.; Okabe, T.; Kiyono, H.; Yamauchi, Y.; Sato, R. Organoid-Derived Intestinal Epithelial Cells Are a Suitable Model for Preclinical Toxicology and Pharmacokinetic Studies. iScience 2022, 25, 104542.

[97] Puzan, M.; Hosic, S.; Ghio, C.; Koppes, A. Enteric Nervous System Regulation of Intestinal Stem Cell Differentiation and Epithelial Monolayer Function. Sci Rep 2018, 8, 6313.

[98] Altay, G.; Larrañaga, E.; Tosi, S.; Barriga, F.M.; Batlle, E.; Fernández-Majada, V.; Martínez, E. Self-Organized Intestinal Epithelial Monolayers in Crypt and Villus-like Domains Show Effective Barrier Function. Sci Rep 2019, 9, 10140.

[99] Hoffmann, P.; Schnepel, N.; Langeheine, M.; Kunnemann, K.; Grassl, G.A.; Brehm, R.; Seeger, B.; Mazzuoli-Weber, G.; Breves, G. Intestinal Organoid-Based 2D Monolayers Mimic Physiological and Pathophysiological Properties of the Pig Intestine. PLoS One 2021, 16, e0256143.

[100] Kawasaki, M.; Ambrosini, Y.M. Accessible Luminal Interface of Bovine Rectal Organoids Generated from Cryopreserved Biopsy Tissues. PLoS One 2024, 19, e0301079.

[104] Felsenthal, N.; Vignjevic, D.M. Stand by Me: Fibroblasts Regulation of the Intestinal Epithelium during Development and Homeostasis. Current Opinion in Cell Biology. 2022, 78, 102116.

[105] Onozato, D.; Ogawa, I.; Kida, Y.; Mizuno, S.; Hashita, T.; Iwao, T.; Matsunaga, T. Generation of Budding-Like Intestinal Organoids from Human Induced Pluripotent Stem Cells. J Pharm Sci 2021, 110, 2637-2650.

[106] Sugimoto, S.; Kobayashi, E.; Fujii, M.; Ohta, Y.; Arai, K.; Matano, M.; Ishikawa, K.; Miyamoto, K.; Toshimitsu, K.; Takahashi, S.; et al. An Organoid-Based Organ-Repurposing Approach to Treat Short Bowel Syndrome. Nature 2021, 592, 99-104.

[109] Pentinmikko, N.; Lozano, R.; Scharaw, S.; Andersson, S.; Englund, J.I.; Castillo-Azofeifa, D.; Gallagher, A.; Broberg, M.; Song, K.Y.; Carvajal, A.S.; et al. Cellular Shape Reinforces Niche to Stem Cell Signaling in the Small Intestine. Sci Adv 2022, 8, eabm1847.

[110] Yavitt, F.M.; Kirkpatrick, B.E.; Blatchley, M.R.; Speckl, K.F.; Mohagheghian, E.; Moldovan, R.; Wang, N.; Dempsey, P.J.; Anseth, K.S. In Situ Modulation of Intestinal Organoid Epithelial Curvature through Photoinduced Viscoelasticity Directs Crypt Morphogenesis. Sci Adv 2023, 9, eadd5668.

[111] Baghdadi, M.B.; Houtekamer, R.M.; Perrin, L.; Rao-Bhatia, A.; Whelen, M.; Decker, L.; Bergert, M.; Pérez-Gonzàlez, C.; Bouras, R.; Gropplero, G.; et al. PIEZO-Dependent Mechanosensing Is Essential for Intestinal Stem Cell Fate Decision and Maintenance. Science 2024, 386, eadj7615.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript by Takahashi and Takase provides a systematic review of intestinal stem cell niches, the mechanistic insights gained from ISCs, and model systems developed to study the gut from ISCs. The manuscript is well-written with helpful illustrations to accompany the text. Some suggestions that could improve the manuscript further are given below:

1) Page 5, last paragraph. Consider revising this sentence that ends in a question to be rewritten as a statement for better consistent writing style: How do Foxl1+ mesenchymal cells control ISC self-renewal through these complex signaling pathways?

2) Page 8, 3.1 subitle - Typo for 3D - please revise to follow similar format as 3.2 subtitle. 

3) While this is briefly touched upon in both 3.1 and 3.2 sections, it would be useful to have a separate section 3.3 comparing 2D vs. 3D models. It is suggested that the authors include a detailed discussion on existing technologies, current challenges, and anticipated future advancements and applications for 2D vs. 3D models. Are there emerging new technologies (for instance, organ-on-chip, 3D bioprinting, etc.)? This could also be included to provide a thorough insight on the model systems.

4) In Conclusions section, please provide a detailed perspective of challenges for therapeutic translation.

Author Response

Comments and Suggestions for Authors

The manuscript by Takahashi and Takase provides a systematic review of intestinal stem cell niches, the mechanistic insights gained from ISCs, and model systems developed to study the gut from ISCs. The manuscript is well-written with helpful illustrations to accompany the text. Some suggestions that could improve the manuscript further are given below:

Response: Thank you very much for your positive comments.

 

• Page 5, last paragraph. Consider revising this sentence that ends in a question to be rewritten as a statement for better consistent writing style: How do Foxl1+ mesenchymal cells control ISC self-renewal through these complex signaling pathways?

Response: We changed the question sentence to the normal sentence as follows.

Foxl1+ mesenchymal cells control ISC self-renewal through these complex signaling pathways.

 

• Page 8, 3.1 subitle - Typo for 3D - please revise to follow similar format as 3.2 subtitle. 

Response: Accordingly, we corrected as “3D” and revised to follow similar format as 3.2 subtitle as “3.1 Three-dimensional (3D) intestinal organoids.”

 

• While this is briefly touched upon in both 3.1 and 3.2 sections, it would be useful to have a separate section 3.3 comparing 2D vs. 3D models. It is suggested that the authors include a detailed discussion on existing technologies, current challenges, and anticipated future advancements and applications for 2D vs. 3D models. Are there emerging new technologies (for instance, organ-on-chip, 3D bioprinting, etc.)? This could also be included to provide a thorough insight on the model systems.

Response: Thank you for your constructive comment. We expanded Section 3 and described a detailed discussion on existing technologies, current challenges, and anticipated future advancements and applications for 2D vs. 3D models in subsection 3.2 and “conclusions and future directions” as follows.

 

• Three-dimensional (3D) intestinal organoids

We added a new paragraph and references as follows.

“As the emergence of cutting-edge technologies including 3D bioprinting or microfluidic chips raises the prospect of closely mimicking the microenvironment [87], current research frequently focuses on combinations of IECs or mesenchymal cells. Multi-organ chips containing vascularization and immunization that connect multiple organ components appear to be better models for studying drug transport and metabolism [88,89].“

(References)

[87] Radhakrishnan, J.; Varadaraj, S.; Dash, S.K.; Sharma, A.; Verma, R.S. Organotypic cancer tissue models for drug screening: 3D constructs, bioprinting and microfluidic chips. Drug Discov. Today 2020, 25, 879–890.

[88] Shirure, V.S.; Hughes, C.C.W.; George, S.C. Engineering vascularized organoid-on-a-chip models. Annu. Rev. Biomed. Eng. 2021, 23, 141–167.

[89] Schafer, S.T.; Mansour, A.A.; Schlachetzki, J.C.M.; Pena, M.; Ghassemzadeh, S.; Mitchell, L.; Mar, A.; Quang, D.; Stumpf, S.; Ortiz, I.S.; et al. An in vivo neuroimmune organoid model to study human microglia phenotypes. Cell 2023, 186, 2111-2126.e2120.

 

• Two-dimensional (2D) epithelial monolayers

We rewritten Subsection 3.2 profoundly as follows.

‘The development of the 3D intestinal organoid culture method has significantly advanced our understanding of ISCs and their niche environment. However, it should be noted that 3D organoids are not a universal experimental model for intestinal research. One major limitation is the orientation of epithelial polarity. In 3D organoids, the apical surface of epithelial cells, which interacts with luminal factors such as dietary components and microbes, faces inward toward the organoid lumen, rendering direct access to this interface experimentally challenging. Additionally, these structures lack distinct villus formations, which are critical for modeling intestinal physiology. To overcome these constraints, various experimental approaches have been explored. For example, methods of injecting substances into the lumen of a 3D organoid or turning the organoid inside out have been reported [90,91]. However, these methods are difficult to perform and inefficient. Additionally, monolayer cultures derived from human colorectal cancer cell lines (Caco-2) have been utilized as analytical systems [92,93]. However, Caco-2 monolayers have been reported to exhibit gene expression and physiological characteristics distinct from those of the native small intestinal epithelium [93,94]. Given these challenges, recent research has focused on developing 2D epithelial monolayers derived from 3D intestinal organoids as an alternative experimental system to overcome these limitations.

The generation of 2D small intestinal epithelial monolayers has been attempted in multiple species, including humans, mice, pigs, and bovines [94-100]. In particular, for human-derived monolayers, several research groups have reported methods using 3D intestinal organoids or iPSC-derived IECs [94-96]. In contrast, generating 2D monolayers from mouse 3D intestinal organoids has been challenging due to the rapid turnover of IECs. In a few cases, methods requiring primary tissue (myofibroblast or enteric nervous system)-derived culture medium or long culture periods have been reported [97,98]. Recently, we developed a method for efficiently forming 2D epithelial monolayers from murine 3D small intestinal organoids using only commercially available reagents and media [101] (Figure 3A). The 2D epithelial monolayers have stable intercellular junctions and contain ISCs and mature IECs [101] (Figure 3B and C). Moreover, the transepithelial electric resistance values of the monolayer were within the expected physiological range [101,102]. This new method is expected to be useful in physiology and pharmaceutical research.

Additionally, 2D intestinal epithelial monolayers, when combined with various experimental approaches, have the potential to provide novel insights into the mechanisms underlying intestinal epithelial morphogenesis. During development, the intestinal epithelium initially forms a flat structure, which subsequently gives rise to characteristic villus and crypt structures through interactions with surrounding tissues [1,103,104]. Recent studies have reported that human 2D intestinal monolayers, when cultured under ALI conditions or subjected to shear forces via shaking culture, which mimics intestinal luminal flow, can induce the formation of villus-like protrusions [105,106]. The intestinal epithelium with its specialized cell organization is intricately folded into arrays of crypts and villi. While the mechanisms driving villi formation are well described, the processes regulating crypt formation remain largely unknown. Pérez-González and coworkers found that the size of ISC compartment depends on the extracellular-matrix stiffness and endogenous cellular forces [81]. Additionally, computational modelling revealed that crypt shape and force distribution rely on cell surface tensions following cortical actomyosin density. They will clarify crypt morphogenesis using 2D intestinal monolayers in the near future. Eicher and colleagues successfully established a co-culture system by separately differentiating endoderm and mesoderm from human iPS cells to generate gastrointestinal tissue-like structures [107]. More recently, Deguchi and colleagues developed a micro-small intestine system by simultaneously differentiating definitive endoderm and mesoderm from human iPS cells on a microfluidic device that replicates intestinal flow. This system enabled the formation of a 3D small-intestine-like tissue with villus-like epithelium and an aligned mesenchymal layer [108]. However, it has not yet fully recapitulated the mature crypt structure. Analysis systems employing microfluidic devices and stiffness-controlled artificial hydrogels to constrain the shape of 2D monolayers and 3D organoids have provided valuable insights into the mechanical properties of ISCs and their niche during intestinal morphogenesis [109-111]. While none of these experimental models perfectly recapitulate the in vivo situation, they continue to advance our understanding of various aspects of intestinal biology.”

 

We added new references as follows.

[81] Pérez-González, C.; Ceada1, G.; Greco, F.; Matejčić1, M.; Gómez-González, M.; Castro1, N.; Menendez, A.; Kale, S.; Krndija, D.; Clark, A.G.; Mechanical compartmentalization of the intestinal organoid enables crypt folding and collective cell migration. Nat. Cell Biol. 2021, 23, 745–757.

[90] Wilson, S.S.; Tocchi, A.; Holly, M.K.; Parks, W.C.; Smith, J.G.A. Small Intestinal Organoid Model of Non-Invasive Enteric Pathogen-Epithelial Cell Interactions. Mucosal Immunol 2015, 8, 352–361.

[91] Co, J.Y.; Margalef-Català, M.; Li, X.; Mah, A.T.; Kuo, C.J.; Monack, D.M.; Amieva, M.R. Controlling Epithelial Polarity: A Human Enteroid Model for Host-Pathogen Interactions. Cell Rep 2019, 26, 2509-2520.e4.

[92] Sambuy, Y.; De Angelis, I.; Ranaldi, G.; Scarino, M.L.; Stammati, A.; Zucco, F. The Caco-2 Cell Line as a Model of the Intestinal Barrier: Influence of Cell and Culture-Related Factors on Caco-2 Cell Functional Characteristics. Cell Biology and Toxicology. 2005, 21, 1–26.

[93] Sun, H.; Chow, E.C.Y.; Liu, S.; Du, Y.; Pang, K.S. The Caco-2 Cell Monolayer: Usefulness and Limitations. Expert Opinion on Drug Metabolism and Toxicology. 2008, 4, 395–411.

[94] Negoro, R.; Takayama, K.; Kawai, K.; Harada, K.; Sakurai, F.; Hirata, K.; Mizuguchi, H. Efficient Generation of Small Intestinal Epithelial-like Cells from Human IPSCs for Drug Absorption and Metabolism Studies. Stem Cell Reports 2018, 11, 1539–1550.

[95] Sasaki, N.; Miyamoto, K.; Maslowski, K.M.; Ohno, H.; Kanai, T.; Sato, T. Development of a Scalable Coculture System for Gut Anaerobes and Human Colon Epithelium. Gastroenterology 2020, 159, 388-390.e5.

[96] Takahashi, Y.; Noguchi, M.; Inoue, Y.; Sato, S.; Shimizu, M.; Kojima, H.; Okabe, T.; Kiyono, H.; Yamauchi, Y.; Sato, R. Organoid-Derived Intestinal Epithelial Cells Are a Suitable Model for Preclinical Toxicology and Pharmacokinetic Studies. iScience 2022, 25, 104542.

[97] Puzan, M.; Hosic, S.; Ghio, C.; Koppes, A. Enteric Nervous System Regulation of Intestinal Stem Cell Differentiation and Epithelial Monolayer Function. Sci Rep 2018, 8, 6313.

[98] Altay, G.; Larrañaga, E.; Tosi, S.; Barriga, F.M.; Batlle, E.; Fernández-Majada, V.; Martínez, E. Self-Organized Intestinal Epithelial Monolayers in Crypt and Villus-like Domains Show Effective Barrier Function. Sci Rep 2019, 9, 10140.

[99] Hoffmann, P.; Schnepel, N.; Langeheine, M.; Kunnemann, K.; Grassl, G.A.; Brehm, R.; Seeger, B.; Mazzuoli-Weber, G.; Breves, G. Intestinal Organoid-Based 2D Monolayers Mimic Physiological and Pathophysiological Properties of the Pig Intestine. PLoS One 2021, 16, e0256143.

[102] Kawasaki, M.; Ambrosini, Y.M. Accessible Luminal Interface of Bovine Rectal Organoids Generated from Cryopreserved Biopsy Tissues. PLoS One 2024, 19, e0301079.

[104] Felsenthal, N.; Vignjevic, D.M. Stand by Me: Fibroblasts Regulation of the Intestinal Epithelium during Development and Homeostasis. Current Opinion in Cell Biology. 2022, 78, 102116.

[105] Onozato, D.; Ogawa, I.; Kida, Y.; Mizuno, S.; Hashita, T.; Iwao, T.; Matsunaga, T. Generation of Budding-Like Intestinal Organoids from Human Induced Pluripotent Stem Cells. J Pharm Sci 2021, 110, 2637-2650.

[106] Sugimoto, S.; Kobayashi, E.; Fujii, M.; Ohta, Y.; Arai, K.; Matano, M.; Ishikawa, K.; Miyamoto, K.; Toshimitsu, K.; Takahashi, S.; et al. An Organoid-Based Organ-Repurposing Approach to Treat Short Bowel Syndrome. Nature 2021, 592, 99-104.

[109] Pentinmikko, N.; Lozano, R.; Scharaw, S.; Andersson, S.; Englund, J.I.; Castillo-Azofeifa, D.; Gallagher, A.; Broberg, M.; Song, K.Y.; Carvajal, A.S.; et al. Cellular Shape Reinforces Niche to Stem Cell Signaling in the Small Intestine. Sci Adv 2022, 8, eabm1847.

[110] Yavitt, F.M.; Kirkpatrick, B.E.; Blatchley, M.R.; Speckl, K.F.; Mohagheghian, E.; Moldovan, R.; Wang, N.; Dempsey, P.J.; Anseth, K.S. In Situ Modulation of Intestinal Organoid Epithelial Curvature through Photoinduced Viscoelasticity Directs Crypt Morphogenesis. Sci Adv 2023, 9, eadd5668.

[111] Baghdadi, M.B.; Houtekamer, R.M.; Perrin, L.; Rao-Bhatia, A.; Whelen, M.; Decker, L.; Bergert, M.; Pérez-Gonzàlez, C.; Bouras, R.; Gropplero, G.; et al. PIEZO-Dependent Mechanosensing Is Essential for Intestinal Stem Cell Fate Decision and Maintenance. Science 2024, 386, eadj7615.

 

“Conclusions and future directions”

“For instance, the technique for conversion from colon organoids into small intestinal tissues is transformative for the development of tissue engineering applications for diseases such as short bowel syndrome [106]. Additionally, many intestinal diseases show changes in crypt morphology including inflammatory bowel disease and irritable bowel disease. These diseases represent an important health burden affecting a significant percentage of the population. The understanding of the mechanisms that regulate crypt malformations in the intestinal stem cell niche may provide critical information to progress in disease treatment and drug development.

                 We discussed ISC-derived organoid culture including their methodology, application, and weakness of intestinal organoids and engineered organoids compared to cell lines. The appropriate applications, advantages and disadvantages of the model have been articulated in Section 3. The development of 2D epithelial monolayers as well as intestinal 3D organoids provides an unprecedented tool for humans to study diseases. By combining microfluidic technology, innovative biological support materials such as extracellular matrices, automated detection methods using AI technologies, we have a more faithful promise that intestinal organoid technology will greatly accelerate the drug discovery process for intestinal diseases and the innovation of treatment methods.

”

We added a new reference as follows.

[106] Sugimoto, S.; Kobayashi, E.; Fujii, M.; Ohta, Y.; Arai, K.; Matano, M.; Ishikawa, K.; Miyamoto, K.; Toshimitsu, K.; Takahashi, S.; et al. An Organoid-Based Organ-Repurposing Approach to Treat Short Bowel Syndrome. Nature 2021, 592, 99-104.

 

• In Conclusions section, please provide a detailed perspective of challenges for therapeutic translation.

Response: According to the reviewer’s comment, we added new sentences for a detailed perspective of challenge for therapeutic translation in “Conclusions and future directions”. Please see our response to Comment 3.

Reviewer 4 Report

Comments and Suggestions for Authors

This comprehensive review summarizes knowledge about the stem cell niche with a focus on the small intestine. Data from mouse and human are assessed provide a picture of the current knowledge with regards to stem cell dynamics and how the niche interplays with these dynamics. Additionally, findings using organoid cultures are integrated. This is a well rounded review, well conceived and researched.

Author Response

Comments and Suggestions for Authors

This comprehensive review summarizes knowledge about the stem cell niche with a focus on the small intestine. Data from mouse and human are assessed provide a picture of the current knowledge with regards to stem cell dynamics and how the niche interplays with these dynamics. Additionally, findings using organoid cultures are integrated. This is a well rounded review, well conceived and researched.

Response: Thank you very much for your positive comment.

Reviewer 5 Report

Comments and Suggestions for Authors

Peer-review report organoids-3419721

Original manuscript title: The intestinal stem cell niche: generation and utilization of intestinal organoids

Overview

This narrative revision outlines the background of intestinal stem cells (ISC) and the niche cells (including epithelial and mesenchymal cells) with a critical role in ISC homeostasis.  Both epithelial and mesenchymal cells provide a wide array of factors involved in the regulation of proliferation, maturation and differentiation of ISC toward terminally differentiated cells including enterocytes and goblet cells. Of noticing,  a very intriguing phenomenon is the dedifferentiation of epithelial cells located at niche such as Paneth cells. This background contextualizes the in vitro cells cultures of organoids based on intestinal cells.

Highlights

Study provides concise and information about basic knowledge of ISC as foundation of organoid cell cultures with potential physiologic and pharmaceutical applications.  

Minor points

1. Clarify please briefly the role of Lgr5 in up- and/or downmodulation of ISC proliferation? （Line 51）

2. Clarify please briefly the role of Wnt/beta catenin in upmodulation of ISC proliferation?（Line 87）

3. It is advisable to depict signal pathways in this Figure. Authors may decide which factors, aside of Lgr5+ and wnt,  involved in signal pathways worth to depict in this figure.（Line 98 ）

Author Response

Overview

This narrative revision outlines the background of intestinal stem cells (ISC) and the niche cells (including epithelial and mesenchymal cells) with a critical role in ISC homeostasis.  Both epithelial and mesenchymal cells provide a wide array of factors involved in the regulation of proliferation, maturation and differentiation of ISC toward terminally differentiated cells including enterocytes and goblet cells. Of noticing,  a very intriguing phenomenon is the dedifferentiation of epithelial cells located at niche such as Paneth cells. This background contextualizes the in vitro cells cultures of organoids based on intestinal cells.

 

Highlights

Study provides concise and information about basic knowledge of ISC as foundation of organoid cell cultures with potential physiologic and pharmaceutical applications. 

Response: Thank you very much for your positive comments.

 

Minor points

1. Clarify please briefly the role of Lgr5 in up- and/or downmodulation of ISC proliferation? （Line 51）

Response: According to the reviewer’s comment, we added a new sentence in line 54 as follows.

“Lgr5 is necessary to ensure proper cell turnover and to confine cell division to crypts to avoid malignant overgrowth.”

 

2. Clarify please briefly the role of Wnt/beta catenin in upmodulation of ISC proliferation?（Line 87）

Response: Thank you for your comment. Accordingly, we added a new sentence in line 87 and new references as follows.

“Genetic and pharmacological evidence indicates that canonical Wnt/beta-catenin signaling is a pivotal role for intestinal homeostasis and ISC proliferation [32-36].”

(New references)

[32] van Es, J.H.; Haegebarth, A.; Kujala, P.; Itzkovitz, S.; Koo, B-K.; Boj, S.F.; Korving, J.; van den Born, M.; van Oudenaarden, A.; Robine, S.; et al. A critical role for the Wnt effector Tcf4 in adult intestinal homeostatic self-renewal. Mol. Cell Biol. 2012, 32, 1918–1927.

[33] Kabiri, Z.; Greicius, G.; Madan, B.; Biechele, S.; Zhong, Z.; Zaribafzadeh, H.; Edison.; Aliyev, J.; Wu, Y.; Bunte, R.; et al. Stroma provides an intestinal stem cell niche in the absence of epithelial Wnts. Development 2014, 141, 2206-2215.

[34] San Roman, A.K.; Jayewickreme, C.D.; Murtaugh, L.C.; Ramesh A. Shivdasani, R.A. Wnt secretion from epithelial cells and subepithelial myofibroblasts is not required in the mouse intestinal stem cell niche in vivo. Stem Cell Rep. 2014, 2, 127–134.

[35] Kuhnert, F.; Davis, C.R.; Wang, H-T.; Chu, P.; Lee, M.; Yuan, J.; Nusse, R.; Kuo, C.J. Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1. Proc Natl Acad Sci USA 2004, 101, 266–271.

[36] Pinto, D.; Gregorieff, A.; Begthel, H.; Clevers, H. Canonical Wnt signals are essential for homeostasis of the intestinal epithelium. Genes Dev. 2003, 17, 1709–1713.

 

3. It is advisable to depict signal pathways in this Figure. Authors may decide which factors, aside of Lgr5+ and wnt,  involved in signal pathways worth to depict in this figure.（Line 98 ）

Response: According to the reviewer’s comment, we added signaling pathways in Figure 2.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The revised manuscript by Takahashi and Takase is improved but there are still some issues.

Comments: 

1. The Abstract should be revised to reflect the manuscript contents, and it should be consistent with the title. Currently, the Abstract is written about ISCs and the niche, but it doesn’t include any information on organoids or monolayers. The Abstract should summarize the whole content of the paper.
2. The following two sentences are contradictory, which creates confusion. They should be revised for clarity (lines 156-159):

“BMP ligands activate BMP receptors, and the signals maintain ISC stemness and promote ISC differentiation through phosphorylated Smad transcription factors [46]. A complex gradient of BMP signaling formed by PDGFα+ cells promotes ISC differentiation rather than maintains ISC stemness.”

3. The use of the term “organogenesis” (line 302), which is defined as “the process by which organs and tissues form from embryonic cells” is the wrong word choice here.
4. Much of the newly added paragraph in section 3 requires references (lines 303-316).
5. The authors have left out some seminal studies on murine 2D IEC organoid monolayers that should be mentioned and cited (PMID 29153987, 24220295).
6. Other studies that first established 2D monolayers from human IEC organoids should also be cited (PMID 25007816, 27562956).
7. The following sentence does not accurately describe the findings of the referenced study and should be revised for accuracy (lines 446-448). Rather, the authors of that study replaced native colonic epithelium with ileum-derived organoids.

“For instance, the technique for conversion from colon organoids into small intestinal tissues is transformative for the development of tissue engineering applications for diseases such as short bowel syndrome [106].”

Author Response

Reviewer 1

 

Comments and Suggestions for Authors

The revised manuscript by Takahashi and Takase is improved but there are still some issues.

Comments: 

1. The Abstract should be revised to reflect the manuscript contents, and it should be consistent with the title. Currently, the Abstract is written about ISCs and the niche, but it doesn’t include any information on organoids or monolayers. The Abstract should summarize the whole content of the paper.

Response: According to the reviewer’s comment, we added new sentences in the abstract as follows.

“Intestinal organoids originate from a group of crypt base ISCs. These organoids possess a three-dimensional (3D) cell structure made up of the lumen facing inward. Therefore, 3D intestinal organoids are often digested and seeded in two-dimensional (2D) to form confluent organoid monolayers.”

“Here, we not only review our current understanding of ISC niches with a focus on systems that are well-characterized at the cellular and mechanistic levels. But also, we summarize the current applications of intestinal organoids.”

 

1. The following two sentences are contradictory, which creates confusion. They should be revised for clarity (lines 156-159):

“BMP ligands activate BMP receptors, and the signals maintain ISC stemness and promote ISC differentiation through phosphorylated Smad transcription factors [46]. A complex gradient of BMP signaling formed by PDGFα+ cells promotes ISC differentiation rather than maintains ISC stemness.”

Response: To avoid contradiction between two sentences, we rewritten the sentence as follows.

“Graded BMP signaling formed by PDGFα+ cells in the intestinal crypt-villus axis guides ISC differentiation and stemness maintenance.

 

1. The use of the term “organogenesis” (line 302), which is defined as “the process by which organs and tissues form from embryonic cells” is the wrong word choice here.

Response: Accordingly, we changed “organogenesis” to “organoid formation”.

 

1. Much of the newly added paragraph in section 3 requires references (lines 303-316).

Response: Accordingly, we added new references in section 3 as follows.

[81] Santos, A.J.M.; Lo, Y-H.; Mah, A.T.; Kuo, C.J. The intestinal stem cell niche: homeostasis and adaptations. Trends Cell Biol. 2018, 28, 1062-1078.

[82] Spradling, A.; Drummond-Barbosa, D.; Kai, T. Stem cells find their niche. Nature 2001, 414, 98-104.

[83] Elosegui-Artola, A. The extracellular matrix viscoelasticity as a regulator of cell and tissue dynamics. Curr. Opin. Cell Biol. 2021, 72, 10-18.

[84] Chaudhuri, O.; Cooper-White, J.; Janmey, P.A.; Shenoy, V.B. Effects of extracellular matrix viscoelasticity on cellular behaviour. Nature 2020, 584, 535-546.

[85] Briscoe, J.; Small, S. Morphogen rules: design principles of gradient-mediated embryo patterning. Development 2015, 142, 3996-4009.

[86] Romanova-Michaelides, M.; Hadjivasiliou, Z.; Aguilar-Hidalgo, D.; Basagiannis, D.; Seum, C.; Dubois, M.; Jülicher, F.; Gonzalez-Gaitan, M. Morphogen gradient scaling by recycling of intracellular Dpp. Nature 2022, 602, 287-293.

[87] Kicheva, A.; Bollenbach, T.; Wartlick, O.; Jülicher, F.; Gonzalez-Gaitan, M. Investigating the principles of morphogen gradient formation: from tissue to cells. Curr. Opin. Genet. Dev. 2012, 22, 527-532.

 

1. The authors have left out some seminal studies on murine 2D IEC organoid monolayers that should be mentioned and cited (PMID 29153987, 24220295).
2. Other studies that first established 2D monolayers from human IEC organoids should also be cited (PMID 25007816, 27562956).

Response: According to the reviewer’s comments, we added new sentences and references as follows.

“The generation of 2D small intestinal epithelial monolayers has been attempted in multiple species, including humans, mice, pigs, and bovines [101-111]. The utility of these 2D monolayers has been demonstrated in several studies, particularly in IgA transcytosis and pathogen-epithelial interactions [102, 103]. In particular, for human-derived monolayers, several research groups have reported methods using 3D intestinal organoids or iPSC-derived IECs [101, 103-107]. In contrast, generating 2D monolayers from mouse 3D intestinal organoids has been challenging due to the rapid turnover of IECs. In some cases, methods have been reported that utilize 3D organoids derived from the colon, where IEC turnover is slower, or directly from living intestinal epithelium to produce monolayers [102, 105]. Additionally, approaches that require primary tissue (myofibroblast or enteric nervous system)-derived culture medium or long culture periods have also been reported [108, 109]. Recently, we developed a method for efficiently forming 2D epithelial monolayers from murine 3D small intestinal organoids using only commercially available reagents and media [112] (Figure 3A). The 2D epithelial monolayers have stable inter-cellular junctions and contain ISCs and mature IECs [112] (Figure 3B and C). Moreover, the transepithelial electric resistance values of the monolayer were within the expected physiological range [112,113]. This new method is expected to be useful in physiology and pharmaceutical research.”

 

[102]. Moon, C.; Vandussen, K.L.; Miyoshi, H.; Stappenbeck, T.S. Development of a Primary Mouse Intestinal Epithelial Cell Monolayer Culture System to Evaluate Factors That Modulate IgA Transcytosis. Mucosal Immunol. 2014, 7, 818-828.

[103]. Ettayebi, K.; Crawford, S.E.; Murakami, K.; Broughman, J.R.; Karandikar, U.; Tenge, V.R.; Neill, F.H.; Blutt, S.E.; Zeng, X.L.; Qu, L.; et al. Replication of Human Noroviruses in Stem Cell-Derived Human Enteroids. Science 2016, 353, 1387-1393.

[104]. VanDussen, K. L.; Marinshaw, J.M.; Shaikh, N.; Miyoshi, H.; Moon, C.; Tarr, P.I.; Ciorba, M.A.; Stappenbeck, T.S. Development of an Enhanced Human Gastrointestinal Epithelial Culture System to Facilitate Patient-Based Assays. Gut 2015, 64, 911-920.

[105]. Kozuka, K.; He, Y.; Koo-McCoy, S.; Kumaraswamy, P.; Nie, B.; Shaw, K.; Chan, P.; Leadbetter, M.; He, L.; Lewis, J.G.; et al. Development and Characterization of a Human and Mouse Intestinal Epithelial Cell Monolayer Platform. Stem Cell Rep. 2017, 9, 1976-1990.

 

1. The following sentence does not accurately describe the findings of the referenced study and should be revised for accuracy (lines 446-448). Rather, the authors of that study replaced native colonic epithelium with ileum-derived organoids.

“For instance, the technique for conversion from colon organoids into small intestinal tissues is transformative for the development of tissue engineering applications for diseases such as short bowel syndrome [106].”

Response: According to the reviewer’s comment, we change sentences as follows.

“For instance, the technique for conversion from native colonic epithelium into small intestinalized colon using ileum-derived organoids is transformative for the development of tissue engineering applications for diseases such as short bowel syndrome [112].”