- Email: [email protected]
Think About the Environment: Cellular Reprogramming by the Extracellular Matrix
Cell Stem Cell
Previews Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region. Nat. Biotechnol. 25, 1315–1321. Jojola, S., Witmer, G., and Nolte, D. (2005). Nutria: An Invasive Rodent Pest or Valued Resource? Proceedings of the 11th Wildlife Damage Management Conference 110.
Kusumbe, A.P., Ramasamy, S.K., and Adams, R.H. (2014). Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature 507, 323–328. €e, Kusumbe, A.P., Ramasamy, S.K., Itkin, T., Ma M.A., Langen, U.H., Betsholtz, C., Lapidot, T., and Adams, R.H. (2016). Age-dependent modula-
tion of vascular niches for haematopoietic stem cells. Nature 532, 380–384. Sipkins, D.A., Wei, X., Wu, J.W., Runnels, J.M., Coˆte´, D., Means, T.K., Luster, A.D., Scadden, D.T., and Lin, C.P. (2005). In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment. Nature 435, 969–973.
Think About the Environment: Cellular Reprogramming by the Extracellular Matrix David J. Huels1,2 and Jan Paul Medema1,2,* 1Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine (CEMM) and Cancer Center Amsterdam, Academic Medical Center, 1105AZ Amsterdam, the Netherlands 2Oncode Institute, Academic Medical Center, 1105AZ Amsterdam, the Netherlands *Correspondence: [email protected] https://doi.org/10.1016/j.stem.2017.12.006
In this issue of Cell Stem Cell, Yui et al. (2018) show how tissue regeneration is driven by changes in the microenvironment. During intestinal regeneration, the epithelium is reprogrammed into a fetal state by an altered extracellular matrix (ECM), which is dependent on YAP/TAZ activation. The adult intestinal epithelium is continuously renewing, and the production of epithelial cells is driven by proliferative stem cells that reside at the bottom of the crypt. However, the epithelium also has the capability to repair itself after tissue damage in a process that is characterized by increased Wnt signaling and regrowth of entire crypts involving distinct cells that then function as stem cells (Blanpain and Fuchs, 2014). In mice, this process can be studied using administration of dextran sulfate sodium (DSS) that induces inflammation of the colon. However, the exact mechanism during tissue repair still remains elusive. In recent years, it was shown that intestinal crypts grown in vitro can be transplanted into damaged colon and regrow the intestinal epithelium (Yui et al., 2012). In order to reach the goal of transplanting intestinal crypts as a therapeutic approach, similar to the spectacular transplantation of the skin that was recently reported (Hirsch et al., 2017), we need to better understand the general mechanisms that are involved during tissue repair. In the current study in this issue of Cell Stem Cell, Yui et al. (2018) analyzed cellular changes during intestinal repair
after DSS treatment. The authors identified that the regenerating epithelium is marked by increased expression of the cell surface marker Sca1/Ly6a. This marker is expressed in the fetal intestine, but normally absent in adult epithelium (Fordham et al., 2013). With this marker at hand, the authors purified regenerating epithelium and revealed a substantial overlap in gene expression with the fetal intestine, suggesting a rewiring of these cells to a more embryonic state. The expression of secretory cell linage markers, but also intestinal stem cell markers, is reduced in Sca1+ cells. However, these cells retain stem cell capacity as they readily form organoids in vitro. Accompanied by these transcriptional changes were alterations in the microenvironment and in cellular proteins that sense the microenvironment. Increased collagen I depositions were found specifically around repairing crypts, which coincided with the activation of the collagen-sensing signal transduction cascade that involves FAK and Src and the activation of the transcription factor YAP (Figure 1A). The importance of this intimate connection between the regenerating epithelium
and the altered environment was further demonstrated by inhibition of FAK and Src that showed impaired repair following damage and a reduction in YAP+ cells, in line with previous reports (Ashton et al., 2010). The authors then queried whether the observed changes in the environment would be sufficient to reprogram the intestinal epithelium. To answer this question, they used the well-established organoid culture. This 3D culture allowed the growth of intestinal crypts in vitro containing intestinal stem cells as well as differentiated cell types, but also provided a setup in which to analyze the impact of changing the environment when the extracellular matrix (ECM) components were altered from mainly laminin and collagen IV (Matrigel) to collagen I. Transcriptional analysis showed that collagen I specifically resulted in the induction of genes that were also enriched in the regenerating epithelium and in the fetal intestine. In addition, loss of polarization and YAP activation were also observed in these organoids, similar to what was seen in the regenerating tissue in vivo. This indicates that changes in the ECM, specifically collagen I, are
Cell Stem Cell 22, January 4, 2018 ª 2017 Elsevier Inc. 7
Cell Stem Cell
Previews A
regenerating crypt
changed Extracellular Matrix
Tissue Damage
crypt under homeostasis
Inflammation
Sca1+ cell
normal extracellular matrix
Sca1+ cell with nuclear YAP
changed extracellular matrix with collagen I
Fetal Intestine
normal colon crypt cell
B
in matrigel + WNT
changed Extracellular Matrix
in collagen + WNT
Figure 1. The Extracellular Matrix Induces Cellular Reprogramming (A) The regenerating crypt is similar to the fetal intestinal epithelium. Upon damage, the normal crypt undergoes dramatic changes and expresses genes that are specifically found in the fetal intestine (e.g., Sca1+). This is accompanied by changes in the extracellular matrix, especially an increase in collagen I. (B) The extracellular matrix induces changes in cell fate. Organoids from crypts were either cultured in Matrigel or in pure collagen I. The collagen-grown organoids express Sca1 and similar genes as expressed by the repairing/fetal intestine.
sufficient to rewire the intestinal cells (Figure 1B). The reprogramming by the ECM in vitro was possible only in combination with Wnt activation (via Wnt3a). Not surprisingly, loss of Apc, which results in deregulated Wnt activity, could compensate for the addition of Wnt3a to the cul8 Cell Stem Cell 22, January 4, 2018
ture medium. Nevertheless, even with deregulated Wnt activity, connection to the ECM is still required as inhibition of the mechano-sensor Rho reduced organoid formation. In other words, increased Wnt activity is required for the organoid growth in collagen, but it cannot compensate for YAP inhibition.
In contrast, overexpression of YAP could compensate for Wnt activation, suggesting a key role for YAP in organoid growth downstream of Wnt, as has been previously suggested (Azzolin et al., 2014). This suggests a model wherein YAP lies downstream of ECM mechanosensing and YAP activation is both
Cell Stem Cell
Previews required and sufficient for the cellular reprogramming. To test this hypothesis in vivo, the authors used mice with conditional lossof-function alleles for YAP and its family member TAZ. Loss of YAP/TAZ under normal homeostasis has no phenotype; however, during DSS-induced damage, a dramatic impairment of tissue repair was observed. Similarly, when intestinal organoids were transplanted into damaged colon, YAP/TAZ was required for successful epithelial repopulation, confirming the in vitro findings. In this light, it is surprising that no difference in transplantation efficacy could be detected between collagen-grown organoids and Matrigel-grown organoids, even though the latter better resemble the regenerating epithelium and already contain active YAP. Although speculative, these data point to a very effective reversion of mature intestinal organoids to a more fetal-like state even upon transplantation. As DSS-damaged intestine shows high levels of collagen I, in contrast to normal intestine, the damaged environment is clearly permissive for such a reversion, but whether this is the underlying reason for repair remains to be elucidated. These findings may partially explain why organoid transplantation protocols rely on tissue damage as this would create ECM alterations, in addition to the necessary space, that allow for cellular reprogramming. This study elegantly shows the strength of the environment to directly shape epithelial cell fate and to promote repair. Combined with recent findings that indi-
cate the marked effects of matrix stiffness on intestinal stem cells and YAP signaling (Gjorevski et al., 2016), this study highlights the importance of the environment in shaping cellular programs. Nevertheless, a clear differentiation should be made between the formation of organoids and the maintenance of established organoids, as the latter appear to grow well when placed into collagen I. In fact, long tubes of fused organoids can be generated in collagen I, which closely resemble the normal intestinal lining (Sachs et al., 2017). This might suggest a specific time-window or cellular state in which the epithelial cells are susceptible to changes induced by the environment. A better understanding of these requirements is clearly needed as environmental changes may also come with a dark side as it has long been known that ECM can dictate tumorigenesis (Bissell and Hines 2011), which may also occur in patients suffering from chronic bowel inflammation. Whether the matrix environment is a key player in this inflammation-associated cancer formation as well remains to be established, although it appears likely. We therefore issue a general warning; think about the environment; it is key to survival and growth.
REFERENCES Ashton, G.H., Morton, J.P., Myant, K., Phesse, T.J., Ridgway, R.A., Marsh, V., Wilkins, J.A., Athineos, D., Muncan, V., Kemp, R., et al. (2010). Focal adhesion kinase is required for intestinal regeneration and tumorigenesis downstream of Wnt/c-Myc signaling. Dev. Cell 19, 259–269.
Azzolin, L., Panciera, T., Soligo, S., Enzo, E., Bicciato, S., Dupont, S., Bresolin, S., Frasson, C., Basso, G., Guzzardo, V., et al. (2014). YAP/TAZ incorporation in the b-catenin destruction complex orchestrates the Wnt response. Cell 158, 157–170. Bissell, M.J., and Hines, W.C. (2011). Why don’t we get more cancer? A proposed role of the microenvironment in restraining cancer progression. Nat. Med. 17, 320–329. Blanpain, C., and Fuchs, E. (2014). Plasticity of epithelial stem cells in tissue regeneration. Science 344, 1242281–1242281. Fordham, R.P., Yui, S., Hannan, N.R.F., Soendergaard, C., Madgwick, A., Schweiger, P.J., Nielsen, O.H., Vallier, L., Pedersen, R.A., Nakamura, T., et al. (2013). Transplantation of expanded fetal intestinal progenitors contributes to colon regeneration after injury. Cell Stem Cell 13, 734–744. Gjorevski, N., Sachs, N., Manfrin, A., Giger, S., Bragina, M.E., Ordo´n˜ez-Mora´n, P., Clevers, H., and Lutolf, M.P. (2016). Designer matrices for intestinal stem cell and organoid culture. Nature 539, 560–564. Hirsch, T., Rothoeft, T., Teig, N., Bauer, J.W., Pellegrini, G., De Rosa, L., Scaglione, D., Reichelt, J., Klausegger, A., Kneisz, D., et al. (2017). Regeneration of the entire human epidermis using transgenic stem cells. Nature 551, 327–332. Sachs, N., Tsukamoto, Y., Kujala, P., Peters, P.J., and Clevers, H. (2017). Intestinal epithelial organoids fuse to form self-organizing tubes in floating collagen gels. Development 144, 1107–1112. Yui, S., Nakamura, T., Sato, T., Nemoto, Y., Mizutani, T., Zheng, X., Ichinose, S., Nagaishi, T., Okamoto, R., Tsuchiya, K., et al. (2012). Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5+ stem cell. Nat. Med. 18, 618–623. Yui, S., Azzolin, L., Maimets, M., Pedersen, M.T., Fordham, R.P., Hansen, S.L., Larsen, H.L., Guiu, J., Alves, M.R.P., Rundsten, C.F., et al. (2018). YAP/TAZ-Dependent Reprogramming of Colonic Epithelium Links ECM Remodeling to Tissue Regeneration. Cell Stem Cell 22, this issue, 35–49.
Cell Stem Cell 22, January 4, 2018 9
Previews Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region. Nat. Biotechnol. 25, 1315–1321. Jojola, S., Witmer, G., and Nolte, D. (2005). Nutria: An Invasive Rodent Pest or Valued Resource? Proceedings of the 11th Wildlife Damage Management Conference 110.
Kusumbe, A.P., Ramasamy, S.K., and Adams, R.H. (2014). Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature 507, 323–328. €e, Kusumbe, A.P., Ramasamy, S.K., Itkin, T., Ma M.A., Langen, U.H., Betsholtz, C., Lapidot, T., and Adams, R.H. (2016). Age-dependent modula-
tion of vascular niches for haematopoietic stem cells. Nature 532, 380–384. Sipkins, D.A., Wei, X., Wu, J.W., Runnels, J.M., Coˆte´, D., Means, T.K., Luster, A.D., Scadden, D.T., and Lin, C.P. (2005). In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment. Nature 435, 969–973.
Think About the Environment: Cellular Reprogramming by the Extracellular Matrix David J. Huels1,2 and Jan Paul Medema1,2,* 1Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine (CEMM) and Cancer Center Amsterdam, Academic Medical Center, 1105AZ Amsterdam, the Netherlands 2Oncode Institute, Academic Medical Center, 1105AZ Amsterdam, the Netherlands *Correspondence: [email protected] https://doi.org/10.1016/j.stem.2017.12.006
In this issue of Cell Stem Cell, Yui et al. (2018) show how tissue regeneration is driven by changes in the microenvironment. During intestinal regeneration, the epithelium is reprogrammed into a fetal state by an altered extracellular matrix (ECM), which is dependent on YAP/TAZ activation. The adult intestinal epithelium is continuously renewing, and the production of epithelial cells is driven by proliferative stem cells that reside at the bottom of the crypt. However, the epithelium also has the capability to repair itself after tissue damage in a process that is characterized by increased Wnt signaling and regrowth of entire crypts involving distinct cells that then function as stem cells (Blanpain and Fuchs, 2014). In mice, this process can be studied using administration of dextran sulfate sodium (DSS) that induces inflammation of the colon. However, the exact mechanism during tissue repair still remains elusive. In recent years, it was shown that intestinal crypts grown in vitro can be transplanted into damaged colon and regrow the intestinal epithelium (Yui et al., 2012). In order to reach the goal of transplanting intestinal crypts as a therapeutic approach, similar to the spectacular transplantation of the skin that was recently reported (Hirsch et al., 2017), we need to better understand the general mechanisms that are involved during tissue repair. In the current study in this issue of Cell Stem Cell, Yui et al. (2018) analyzed cellular changes during intestinal repair
after DSS treatment. The authors identified that the regenerating epithelium is marked by increased expression of the cell surface marker Sca1/Ly6a. This marker is expressed in the fetal intestine, but normally absent in adult epithelium (Fordham et al., 2013). With this marker at hand, the authors purified regenerating epithelium and revealed a substantial overlap in gene expression with the fetal intestine, suggesting a rewiring of these cells to a more embryonic state. The expression of secretory cell linage markers, but also intestinal stem cell markers, is reduced in Sca1+ cells. However, these cells retain stem cell capacity as they readily form organoids in vitro. Accompanied by these transcriptional changes were alterations in the microenvironment and in cellular proteins that sense the microenvironment. Increased collagen I depositions were found specifically around repairing crypts, which coincided with the activation of the collagen-sensing signal transduction cascade that involves FAK and Src and the activation of the transcription factor YAP (Figure 1A). The importance of this intimate connection between the regenerating epithelium
and the altered environment was further demonstrated by inhibition of FAK and Src that showed impaired repair following damage and a reduction in YAP+ cells, in line with previous reports (Ashton et al., 2010). The authors then queried whether the observed changes in the environment would be sufficient to reprogram the intestinal epithelium. To answer this question, they used the well-established organoid culture. This 3D culture allowed the growth of intestinal crypts in vitro containing intestinal stem cells as well as differentiated cell types, but also provided a setup in which to analyze the impact of changing the environment when the extracellular matrix (ECM) components were altered from mainly laminin and collagen IV (Matrigel) to collagen I. Transcriptional analysis showed that collagen I specifically resulted in the induction of genes that were also enriched in the regenerating epithelium and in the fetal intestine. In addition, loss of polarization and YAP activation were also observed in these organoids, similar to what was seen in the regenerating tissue in vivo. This indicates that changes in the ECM, specifically collagen I, are
Cell Stem Cell 22, January 4, 2018 ª 2017 Elsevier Inc. 7
Cell Stem Cell
Previews A
regenerating crypt
changed Extracellular Matrix
Tissue Damage
crypt under homeostasis
Inflammation
Sca1+ cell
normal extracellular matrix
Sca1+ cell with nuclear YAP
changed extracellular matrix with collagen I
Fetal Intestine
normal colon crypt cell
B
in matrigel + WNT
changed Extracellular Matrix
in collagen + WNT
Figure 1. The Extracellular Matrix Induces Cellular Reprogramming (A) The regenerating crypt is similar to the fetal intestinal epithelium. Upon damage, the normal crypt undergoes dramatic changes and expresses genes that are specifically found in the fetal intestine (e.g., Sca1+). This is accompanied by changes in the extracellular matrix, especially an increase in collagen I. (B) The extracellular matrix induces changes in cell fate. Organoids from crypts were either cultured in Matrigel or in pure collagen I. The collagen-grown organoids express Sca1 and similar genes as expressed by the repairing/fetal intestine.
sufficient to rewire the intestinal cells (Figure 1B). The reprogramming by the ECM in vitro was possible only in combination with Wnt activation (via Wnt3a). Not surprisingly, loss of Apc, which results in deregulated Wnt activity, could compensate for the addition of Wnt3a to the cul8 Cell Stem Cell 22, January 4, 2018
ture medium. Nevertheless, even with deregulated Wnt activity, connection to the ECM is still required as inhibition of the mechano-sensor Rho reduced organoid formation. In other words, increased Wnt activity is required for the organoid growth in collagen, but it cannot compensate for YAP inhibition.
In contrast, overexpression of YAP could compensate for Wnt activation, suggesting a key role for YAP in organoid growth downstream of Wnt, as has been previously suggested (Azzolin et al., 2014). This suggests a model wherein YAP lies downstream of ECM mechanosensing and YAP activation is both
Cell Stem Cell
Previews required and sufficient for the cellular reprogramming. To test this hypothesis in vivo, the authors used mice with conditional lossof-function alleles for YAP and its family member TAZ. Loss of YAP/TAZ under normal homeostasis has no phenotype; however, during DSS-induced damage, a dramatic impairment of tissue repair was observed. Similarly, when intestinal organoids were transplanted into damaged colon, YAP/TAZ was required for successful epithelial repopulation, confirming the in vitro findings. In this light, it is surprising that no difference in transplantation efficacy could be detected between collagen-grown organoids and Matrigel-grown organoids, even though the latter better resemble the regenerating epithelium and already contain active YAP. Although speculative, these data point to a very effective reversion of mature intestinal organoids to a more fetal-like state even upon transplantation. As DSS-damaged intestine shows high levels of collagen I, in contrast to normal intestine, the damaged environment is clearly permissive for such a reversion, but whether this is the underlying reason for repair remains to be elucidated. These findings may partially explain why organoid transplantation protocols rely on tissue damage as this would create ECM alterations, in addition to the necessary space, that allow for cellular reprogramming. This study elegantly shows the strength of the environment to directly shape epithelial cell fate and to promote repair. Combined with recent findings that indi-
cate the marked effects of matrix stiffness on intestinal stem cells and YAP signaling (Gjorevski et al., 2016), this study highlights the importance of the environment in shaping cellular programs. Nevertheless, a clear differentiation should be made between the formation of organoids and the maintenance of established organoids, as the latter appear to grow well when placed into collagen I. In fact, long tubes of fused organoids can be generated in collagen I, which closely resemble the normal intestinal lining (Sachs et al., 2017). This might suggest a specific time-window or cellular state in which the epithelial cells are susceptible to changes induced by the environment. A better understanding of these requirements is clearly needed as environmental changes may also come with a dark side as it has long been known that ECM can dictate tumorigenesis (Bissell and Hines 2011), which may also occur in patients suffering from chronic bowel inflammation. Whether the matrix environment is a key player in this inflammation-associated cancer formation as well remains to be established, although it appears likely. We therefore issue a general warning; think about the environment; it is key to survival and growth.
REFERENCES Ashton, G.H., Morton, J.P., Myant, K., Phesse, T.J., Ridgway, R.A., Marsh, V., Wilkins, J.A., Athineos, D., Muncan, V., Kemp, R., et al. (2010). Focal adhesion kinase is required for intestinal regeneration and tumorigenesis downstream of Wnt/c-Myc signaling. Dev. Cell 19, 259–269.
Azzolin, L., Panciera, T., Soligo, S., Enzo, E., Bicciato, S., Dupont, S., Bresolin, S., Frasson, C., Basso, G., Guzzardo, V., et al. (2014). YAP/TAZ incorporation in the b-catenin destruction complex orchestrates the Wnt response. Cell 158, 157–170. Bissell, M.J., and Hines, W.C. (2011). Why don’t we get more cancer? A proposed role of the microenvironment in restraining cancer progression. Nat. Med. 17, 320–329. Blanpain, C., and Fuchs, E. (2014). Plasticity of epithelial stem cells in tissue regeneration. Science 344, 1242281–1242281. Fordham, R.P., Yui, S., Hannan, N.R.F., Soendergaard, C., Madgwick, A., Schweiger, P.J., Nielsen, O.H., Vallier, L., Pedersen, R.A., Nakamura, T., et al. (2013). Transplantation of expanded fetal intestinal progenitors contributes to colon regeneration after injury. Cell Stem Cell 13, 734–744. Gjorevski, N., Sachs, N., Manfrin, A., Giger, S., Bragina, M.E., Ordo´n˜ez-Mora´n, P., Clevers, H., and Lutolf, M.P. (2016). Designer matrices for intestinal stem cell and organoid culture. Nature 539, 560–564. Hirsch, T., Rothoeft, T., Teig, N., Bauer, J.W., Pellegrini, G., De Rosa, L., Scaglione, D., Reichelt, J., Klausegger, A., Kneisz, D., et al. (2017). Regeneration of the entire human epidermis using transgenic stem cells. Nature 551, 327–332. Sachs, N., Tsukamoto, Y., Kujala, P., Peters, P.J., and Clevers, H. (2017). Intestinal epithelial organoids fuse to form self-organizing tubes in floating collagen gels. Development 144, 1107–1112. Yui, S., Nakamura, T., Sato, T., Nemoto, Y., Mizutani, T., Zheng, X., Ichinose, S., Nagaishi, T., Okamoto, R., Tsuchiya, K., et al. (2012). Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5+ stem cell. Nat. Med. 18, 618–623. Yui, S., Azzolin, L., Maimets, M., Pedersen, M.T., Fordham, R.P., Hansen, S.L., Larsen, H.L., Guiu, J., Alves, M.R.P., Rundsten, C.F., et al. (2018). YAP/TAZ-Dependent Reprogramming of Colonic Epithelium Links ECM Remodeling to Tissue Regeneration. Cell Stem Cell 22, this issue, 35–49.
Cell Stem Cell 22, January 4, 2018 9