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Clocking the Pace of Organoid Research
EDITORIAL Clocking the Pace of Organoid Research
O
rganoids are 3-dimensional structures that reflect some of the complex features of a native tissue, making them “organ like.”1,2 Organoids have seen a rapid growth in popularity and are becoming a mainstay in gastrointestinal research, owing in part to the ability of organoids to recapitulate some physiological functions of the tissue from which they are derived. For example, intestinal organoids possess the full complement of epithelial cell types and, unlike most other in vitro systems, provide the cellular heterogeneity of the native intestine. Because organoids represent only part of the native organ (ie, the epithelium) and lack other components such as the immune system, vasculature, and nervous tissue normally present, they are a reductionist system that allows researchers to address targeted questions free of the complex interactions making up the organ. Despite their utility, organoids are not without limitations. For example, because they are grown as 3-dimensional structures in a droplet of extracellular matrix, their culture can be cumbersome, and accessing the apical (luminal) surface of the epithelium is difficult and requires time-consuming and labor-intensive approaches, such as microinjection.3,4 Three-dimensional culture methods also limit use of organoids in established pipelines, such as high-throughput screening and other platforms designed for cells grown as 2-dimensional monolayers. Two new articles published in this issue of Cellular and Molecular Gastroenterology and Hepatology involve the use of organoids as models of normal physiology5 and for the development of strategies that overcome limitations inherent to 3-dimensional culture methods.6 In the first study, Stokes et al5 investigate a role for the circadian clock during intestinal regeneration. It is well appreciated that the circadian clock plays an important role regulating normal physiology and behavior by controlling hormonal fluctuations, sleep patterns, feeding, etc. In this study, the authors demonstrate that the intestinal epithelium displays a BMAL1-dependent circadian rhythm during normal homeostasis and during regeneration. Interestingly, although the epithelium displays a rhythm at steady state and following injury, it is only during injury that mitosis is also synchronized with the epithelial clock. In this study, organoids were used to examine if these observations were intrinsic to the epithelium, or if they were being influenced by extrinsic factors such as the nervous system. Based on these experiments, the authors were able to conclude that the BMAL1-dependent clock was intrinsic to the epithelium, a finding that would have been difficult to delineate in vivo. Mechanistically, the study demonstrated that during injury or inflammation BMAL1 regulates the timing of cell proliferation by regulating tumor necrosis factor expression, which was shown to have a direct effect on proliferation. Additionally, BMAL1 mutant mice did not respond properly to tumor
necrosis factor, suggesting that the clock also regulated the ability of the epithelium to respond to inflammatory stress. In the second study, Wang et al6 tackle the limitations of 3-dimensional (epithelium-only) organoids by developing substrates with mechanical properties that allow colonic stem cells to grow in 2-dimensional culture. On appropriate surfaces, intestinal stem cell colonies grew continuously in culture and could be passaged, induced to differentiate, and transferred back to 3-dimensional culture. Although organoids are typically grown in a droplet of Matrigel (Corning Life Sciences, Corning, NY), an extracellular matrix derived from tumor cells, the authors found that when isolated crypts were placed on top of a Matrigel-coated surface, cells failed to spread and instead grew in clumps. When plated on hard tissue culture plastic coated with a number of different extracellular matrix proteins, crypts adhered but failed to expand and ultimately the cells died. On the other hand, the authors found that when grown on a thick collagen bed (collagen hydrogel) at a precise concentration and thickness, the crypts attached and expanded. Failed attempts to grow crypts at lower concentrations of collagen or Matrigel supported the idea that optimal mechanical properties are required for stem cell expansion, a notion also demonstrated recently in 3-dimensional culture using tunable PEG-hydrogels.7 The Wang et al study represents a major advance for the field, and overcomes 2 major limitations: access and scale. Two-dimensional cultures solve 2 different types of access problems. First, 3-dimensional organoid culture is cumbersome, time consuming, and technically challenging; thus, 2-dimensional cultures will likely be accessable to a wider range of researchers familiar with 2-dimensional culture methods. Second, 2-dimensional culture will allow a significantly simpler system for growing epithelial monolayers with an accessible apical (luminal) surface facilitating a host of physiological studies. Regarding scale, it is perceived that using 2-dimensional culture methods will provide a more straight forward approach to culturing multiple cell lines at once and will also be scalable as an approach to obtain sufficient tissue for analyses such as high-content screening. Taken together, the studies of Stokes et al5 and Wang et al6 exploit the power of organoids to understand how the circadian clock functions during homeostasis and regeneration and to develop improved intestinal stem cell culture systems, respectively. They each represent significant conceptual and technical advances in intestinal epithelial biology. JASON R. SPENCE, PhD Department of Internal Medicine and Department of Cell and Developmental Biology University of Michigan Medical School Ann Arbor, Michigan
Cellular and Molecular Gastroenterology and Hepatology 2017;4:203–204
204
Jason R. Spence
References 1.
2.
3.
4.
5.
Dedhia PH, Bertaux-Skeirik N, Zavros Y, Spence JR. Organoid models of human gastrointestinal development and disease. Gastroenterology 2016;150:1098–1112. Hill DR, Spence JR. Gastrointestinal organoids: understanding the molecular basis of the host-microbe interface. Cell Mol Gastroenterol Hepatol 2017;3:138–149. Leslie JL, Huang S, Opp JS, Nagy MS, Kobayashi M, Young VB, Spence JR. Persistence and toxin production by Clostridium difficile within human intestinal organoids results in disruption of epithelial paracellular barrier function. Infect Immun 2015;83:138–145. McCracken KW, Catá EM, Crawford CM, Sinagoga KL, Schumacher M, Rockich BE, Tsai YH, Mayhew CN, Spence JR, Zavros Y, Wells JM. Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. Nature 2014;516:400–404. Stokes K, Cooke A, Chang H, Weaver DR, Breault DT, Karpowicz P. The circadian clock gene BMAL1 coordinates intestinal regeneration. Cell Mol Gastroenterol Hepatol 2017;4:95–114.
Cellular and Molecular Gastroenterology and Hepatology Vol. 4, No. 1 6.
7.
Wang Y, DiSalvo M, Gunasekara DB, Dutton J, Proctor A, Lebhar MS, Williamson IA, Speer J, Howard RL, Smiddy NM, Bultman SJ, Sims CE, Magness ST, Allbritton NL. Self-renewing monolayer of primary colonic or rectal epithelial cells. Cell Mol Gastroenterol Hepatol 2017;4:165–182. Gjorevski N, Sachs N, Manfrin A, Giger S, Bragina ME, Ordóñez-Morán P, Clevers H, Lutolf MP. Designer matrices for intestinal stem cell and organoid culture. Nature 2016;539:560–564.
Correspondence Address correspondence to: Jason R. Spence, Department of Internal Medicine, University of Michigan Medical School, 109 Zina Pitcher Place, Ann Arbor, Michigan 48109. e-mail: [email protected]. Conflicts of interest The author discloses no conflicts. Most current article
© 2017 The Author. Published by Elsevier Inc. on behalf of the AGA Institute. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 2352-345X http://dx.doi.org/10.1016/j.jcmgh.2017.04.006
O
rganoids are 3-dimensional structures that reflect some of the complex features of a native tissue, making them “organ like.”1,2 Organoids have seen a rapid growth in popularity and are becoming a mainstay in gastrointestinal research, owing in part to the ability of organoids to recapitulate some physiological functions of the tissue from which they are derived. For example, intestinal organoids possess the full complement of epithelial cell types and, unlike most other in vitro systems, provide the cellular heterogeneity of the native intestine. Because organoids represent only part of the native organ (ie, the epithelium) and lack other components such as the immune system, vasculature, and nervous tissue normally present, they are a reductionist system that allows researchers to address targeted questions free of the complex interactions making up the organ. Despite their utility, organoids are not without limitations. For example, because they are grown as 3-dimensional structures in a droplet of extracellular matrix, their culture can be cumbersome, and accessing the apical (luminal) surface of the epithelium is difficult and requires time-consuming and labor-intensive approaches, such as microinjection.3,4 Three-dimensional culture methods also limit use of organoids in established pipelines, such as high-throughput screening and other platforms designed for cells grown as 2-dimensional monolayers. Two new articles published in this issue of Cellular and Molecular Gastroenterology and Hepatology involve the use of organoids as models of normal physiology5 and for the development of strategies that overcome limitations inherent to 3-dimensional culture methods.6 In the first study, Stokes et al5 investigate a role for the circadian clock during intestinal regeneration. It is well appreciated that the circadian clock plays an important role regulating normal physiology and behavior by controlling hormonal fluctuations, sleep patterns, feeding, etc. In this study, the authors demonstrate that the intestinal epithelium displays a BMAL1-dependent circadian rhythm during normal homeostasis and during regeneration. Interestingly, although the epithelium displays a rhythm at steady state and following injury, it is only during injury that mitosis is also synchronized with the epithelial clock. In this study, organoids were used to examine if these observations were intrinsic to the epithelium, or if they were being influenced by extrinsic factors such as the nervous system. Based on these experiments, the authors were able to conclude that the BMAL1-dependent clock was intrinsic to the epithelium, a finding that would have been difficult to delineate in vivo. Mechanistically, the study demonstrated that during injury or inflammation BMAL1 regulates the timing of cell proliferation by regulating tumor necrosis factor expression, which was shown to have a direct effect on proliferation. Additionally, BMAL1 mutant mice did not respond properly to tumor
necrosis factor, suggesting that the clock also regulated the ability of the epithelium to respond to inflammatory stress. In the second study, Wang et al6 tackle the limitations of 3-dimensional (epithelium-only) organoids by developing substrates with mechanical properties that allow colonic stem cells to grow in 2-dimensional culture. On appropriate surfaces, intestinal stem cell colonies grew continuously in culture and could be passaged, induced to differentiate, and transferred back to 3-dimensional culture. Although organoids are typically grown in a droplet of Matrigel (Corning Life Sciences, Corning, NY), an extracellular matrix derived from tumor cells, the authors found that when isolated crypts were placed on top of a Matrigel-coated surface, cells failed to spread and instead grew in clumps. When plated on hard tissue culture plastic coated with a number of different extracellular matrix proteins, crypts adhered but failed to expand and ultimately the cells died. On the other hand, the authors found that when grown on a thick collagen bed (collagen hydrogel) at a precise concentration and thickness, the crypts attached and expanded. Failed attempts to grow crypts at lower concentrations of collagen or Matrigel supported the idea that optimal mechanical properties are required for stem cell expansion, a notion also demonstrated recently in 3-dimensional culture using tunable PEG-hydrogels.7 The Wang et al study represents a major advance for the field, and overcomes 2 major limitations: access and scale. Two-dimensional cultures solve 2 different types of access problems. First, 3-dimensional organoid culture is cumbersome, time consuming, and technically challenging; thus, 2-dimensional cultures will likely be accessable to a wider range of researchers familiar with 2-dimensional culture methods. Second, 2-dimensional culture will allow a significantly simpler system for growing epithelial monolayers with an accessible apical (luminal) surface facilitating a host of physiological studies. Regarding scale, it is perceived that using 2-dimensional culture methods will provide a more straight forward approach to culturing multiple cell lines at once and will also be scalable as an approach to obtain sufficient tissue for analyses such as high-content screening. Taken together, the studies of Stokes et al5 and Wang et al6 exploit the power of organoids to understand how the circadian clock functions during homeostasis and regeneration and to develop improved intestinal stem cell culture systems, respectively. They each represent significant conceptual and technical advances in intestinal epithelial biology. JASON R. SPENCE, PhD Department of Internal Medicine and Department of Cell and Developmental Biology University of Michigan Medical School Ann Arbor, Michigan
Cellular and Molecular Gastroenterology and Hepatology 2017;4:203–204
204
Jason R. Spence
References 1.
2.
3.
4.
5.
Dedhia PH, Bertaux-Skeirik N, Zavros Y, Spence JR. Organoid models of human gastrointestinal development and disease. Gastroenterology 2016;150:1098–1112. Hill DR, Spence JR. Gastrointestinal organoids: understanding the molecular basis of the host-microbe interface. Cell Mol Gastroenterol Hepatol 2017;3:138–149. Leslie JL, Huang S, Opp JS, Nagy MS, Kobayashi M, Young VB, Spence JR. Persistence and toxin production by Clostridium difficile within human intestinal organoids results in disruption of epithelial paracellular barrier function. Infect Immun 2015;83:138–145. McCracken KW, Catá EM, Crawford CM, Sinagoga KL, Schumacher M, Rockich BE, Tsai YH, Mayhew CN, Spence JR, Zavros Y, Wells JM. Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. Nature 2014;516:400–404. Stokes K, Cooke A, Chang H, Weaver DR, Breault DT, Karpowicz P. The circadian clock gene BMAL1 coordinates intestinal regeneration. Cell Mol Gastroenterol Hepatol 2017;4:95–114.
Cellular and Molecular Gastroenterology and Hepatology Vol. 4, No. 1 6.
7.
Wang Y, DiSalvo M, Gunasekara DB, Dutton J, Proctor A, Lebhar MS, Williamson IA, Speer J, Howard RL, Smiddy NM, Bultman SJ, Sims CE, Magness ST, Allbritton NL. Self-renewing monolayer of primary colonic or rectal epithelial cells. Cell Mol Gastroenterol Hepatol 2017;4:165–182. Gjorevski N, Sachs N, Manfrin A, Giger S, Bragina ME, Ordóñez-Morán P, Clevers H, Lutolf MP. Designer matrices for intestinal stem cell and organoid culture. Nature 2016;539:560–564.
Correspondence Address correspondence to: Jason R. Spence, Department of Internal Medicine, University of Michigan Medical School, 109 Zina Pitcher Place, Ann Arbor, Michigan 48109. e-mail: [email protected]. Conflicts of interest The author discloses no conflicts. Most current article
© 2017 The Author. Published by Elsevier Inc. on behalf of the AGA Institute. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 2352-345X http://dx.doi.org/10.1016/j.jcmgh.2017.04.006