AUTHOR'S VIEW Molecular & Cellular Oncology 1:3, e970069; July/August/September 2014; Published with license by Taylor & Francis Group, LLC
Revealing human intestinal stem cell and crypt dynamics Ann-Marie Baker* and Trevor A Graham* Evolution and Cancer Laboratory; Barts Cancer Institute; Barts and the London School of Medicine and Dentistry; Queen Mary University of London; London, UK
Keywords: clonal evolution, crypt life-cycle, human intestine, neutral drift, stem cell
Stem cell and crypt dynamics in the human gut have been remarkably poorly characterized. We used random somatic mutations to trace stem cell lineages in the human intestine and coupled these data with mathematical modeling to infer the in vivo temporal dynamics of human intestinal stem cells.
The application of transgenic lineage tracing technology has led to tremendous advances in our understanding of the in vivo behavior of adult stem cells in model organisms. The stem cell biology of the intestinal crypt is a prime example. Crypts are finger-like invaginations present along the entirety of the intestinal epithelium, and the flux of cells along the crypt (production at the crypt base, migration along the crypt axis, and shedding into the lumen) implies that each crypt is sustained by the clonal expansion, vertical migration, and differentiation of basally located colonic stem cells (See Fig. 1). In mice, the application of transgenic lineage tracing has led to the identification of a set of “marker” genes (such as Lgr51) that are expressed by the basal stem cells. Subsequent mathematical modeling of clone-level data has quantified the continual competition that occurs between stem cells for the retention of a position in the niche.2-4 However, human intestinal stem cell behavior has remained poorly described because transgenic lineage tracing strategies cannot be applied to humans. In our study recently published in Cell Reports,5 we described for the first time a novel method of examining in vivo stem cell behavior within the human colon. We used naturally occurring somatic mutations to trace clonal lineages in the human intestinal crypt, coupled with mathematical modeling to infer the temporal dynamics of stem cell evolution.
To detect somatic mutations we used loss of activity of the mitochondrially-encoded enzyme cytochrome c oxidase (CCO), which can be attributed to an underlying somatic mutation.6 Since a CCO mutation that occurs in a long-lived stem cell will be inherited by all of its progeny, we were able to use CCO loss of activity (CCO¡) to track clonal populations within the colon.7 We examined serial en-face sections throughout the length of human colonic crypts in which CCO¡ clones in each section were visualized using enzyme histochemistry (with CCO¡ cells appearing blue and CCOC cells brown). We then stacked the serial sections to form a reconstruction (or ‘map’) of the crypt in question in which CCO¡ clones can be seen to arise in the crypt base and extend through the crypt in a blue ‘clonal ribbon’ (see Fig. 1). We realized that the change in width of the CCO¡ ribbon along the crypt axis is governed by the division of stem cells at the base. When a CCO¡ stem cell is lost from the niche the ribbon width decreases, and conversely when a CCO¡ stem cell divides and displaces a CCOC stem cell from the niche, the ribbon gets wider. The changes in ribbon width along the crypt axis are effectively a short-term record of events in the stem cell niche, as the distance along the vertical axis of the crypt is proportional to the elapsed time since the cells were generated at the crypt base. Thus, to infer the in vivo behavior of human intestinal stem cells, we quantified
the deviations in CCO¡ ribbon width along the crypt axis and then performed a mathematical analysis of these data, modeling the changes in ribbon width as a random walk. We found that normal human colonic crypts contain a small number of functional stem cells (approximately 5–6), a similar number to the murine crypt.8 Importantly, we showed that increases in ribbon width are balanced by decreases in ribbon width, demonstrating neutral drift of the stem cell population. In addition to analyzing healthy human crypts, we studied tissue from patients with familial adenomatous polyposis (FAP) who carry a germline mutation in one copy of the tumor suppressor gene adenomatous polyposis coli (APC).9 Loss of the remaining copy of APC leads to adenoma development. We found an increase in stem cell number and the loss/replacement rate in FAP adenomas, indicating that the parameters of stem cell competition within the niche are altered. Interestingly, although stem cell dynamics are fundamentally different within FAP adenomatous crypts, they remain governed by a neutral drift process. We recognize that our estimations of stem cell dynamics were based on historical measurements of certain parameters, and that the margin of error of these values will of course impact the accuracy of our calculations. In addition to our estimations of stem cell dynamics, we were also able to exploit CCO mutations to examine the dynamics of crypt division, the process responsible for
© Ann-Marie Baker and Trevor A Graham *Correspondence to: Ann-Marie Baker; Email: [email protected]; Trevor A Graham; Email: [email protected] Submitted: 09/02/2014; Revised: 09/04/2014; Accepted: 09/06/2014 http://dx.doi.org/10.4161/23723548.2014.970069
www.landesbioscience.com
Molecular & Cellular Oncology
e970069-1
Figure 1. Inferring intestinal stem cell dynamics from clonal imprints on the crypt wall. Schematic showing a colonic crypt with a ribbon of cytochrome c oxidase-deficient cells (CCO¡, blue color) migrating from the crypt base to the top of the crypt; this ribbon is a “clonal imprint” on the crypt wall. Changes in the CCO¡ ribbon width (termed “wiggles”) along the crypt axis are evident. We hypothesized that changes in ribbon width represent a record of the dynamics of the stem cells at the crypt base; specifically, that increases in the ribbon width were caused by replacement of a wild-type stem cell (brown) with a CCO¡ stem cell (blue), whereas decreases in the ribbon width were due to loss of a CCO¡ stem cell from the pool. By quantitatively analyzing the changes in ribbon width, we were able to infer the temporal dynamics of stem cell evolution.
early adenoma growth. When a CCO¡ stem cell expands to take over the stem cell niche, the crypt will become entirely CCO¡, and if this crypt then undergoes sequential fission events there will be a ‘patch’ of adjacent CCO¡ crypts. We analyzed the size and distribution of such patches mathematically (using a birth-process model) to determine the crypt division rate. We found that a normal colonic crypt divides once every 30–40 years, and in FAP adenomas this rate increases by an order of
magnitude. This suggests that even in adenomas the fission rate is remarkably slow, a result consistent with the observed slow progression of adenoma to carcinoma.10 In summary, we quantified the size of somatic mutant clones in the human intestine to estimate in vivo stem cell and crypt dynamics in health and disease. We found that stem cell number, stem cell loss/replacement rate, and crypt fission rate are elevated in FAP adenomas, indicating an important role for APC in the
regulation of stem cell and crypt dynamics. Our methodology of mathematically inferring clonal dynamics from static images is readily transferrable to other tissues and tumors and therefore has potential to reveal stem cell biology throughout the human body.
References
449(7165):1003–7; PMID:17934449; http://dx.doi. org/10.1038/nature06196 2. Lopez-Garcia C, Klein AM, Simons BD, Winton DJ. Intestinal stem cell replacement follows a pattern of neutral
drift. Science 2010; 330(6005):822–5; PMID:20929733; http://dx.doi.org/10.1126/science.1196236 3. Ritsma L, Ellenbroek SI, Zomer A, Snippert HJ, de Sauvage FJ, Simons BD, Clevers H, van Rheenen J. Intestinal crypt homeostasis revealed at single-stem-cell
1. Barker N, van Es JH, Kuipers J, Kujala P, van den Born M, Cozijnsen M, Haegebarth A, Korving J, Begthel H, Peters PJ, et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 2007;
e970069-2
Molecular & Cellular Oncology
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Volume 1 Issue 3
level by in vivo live imaging. Nature 2014; 507 (7492):362–5; PMID:24531760; http://dx.doi.org/ 10.1038/nature12972 4. Snippert HJ, van der Flier LG, Sato T, van Es JH, van den Born M, Kroon-Veenboer C, Barker N, Klein AM, van Rheenen J, Simons BD, et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 2010; 143 (1):134–44; PMID:20887898; http://dx.doi.org/ 10.1016/j.cell.2010.09.016 5. Baker AM, Cereser B, Melton S, Fletcher AG, Rodriguez-Justo M, Tadrous PJ, Humphries A, Elia G, McDonald SA, Wright NA, et al. Quantification of crypt and stem cell evolution in the normal and neoplastic human colon. Cell Rep 2014; 8:940–7; PMID:25127143; http://dx.doi.org/10.1016/j. celrep.2014.07.019
www.landesbioscience.com
6. Taylor RW, Barron MJ, Borthwick GM, Gospel A, Chinnery PF, Samuels DC, Taylor GA, Plusa SM, Needham SJ, Greaves LC, et al. Mitochondrial DNA mutations in human colonic crypt stem cells. J Clin Invest 2003; 112(9):1351–60; PMID:14597761; http://dx.doi.org/10.1172/JCI19435 7. Fellous TG, McDonald SA, Burkert J, Humphries A, Islam S, De-Alwis NM, Gutierrez-Gonzalez L, Tadrous PJ, Elia G, Kocher HM, et al. A methodological approach to tracing cell lineage in human epithelial tissues. Stem Cells 2009; 27(6):1410–20; PMID:19489031; http://dx.doi.org/10.1002/stem.67 8. Kozar S, Morrissey E, Nicholson AM, van der Heijden M, Zecchini HI, Kemp R, Tavare S, Vermeulen L, Winton DJ, et al. Continuous clonal labeling reveals small numbers of functional stem cells in intestinal crypts and adenomas. Cell Stem Cell 2013; 13(5):626–
Molecular & Cellular Oncology
33; PMID:24035355; http://dx.doi.org/10.1016/j. stem.2013.08.001 9. Miyoshi Y, Ando H, Nagase H, Nishisho I, Horii A, Miki Y, Mori T, Utsunomiya J, Baba S, Petersen G, et al. Germ-line mutations of the APC gene in 53 familial adenomatous polyposis patients. Proc Natl Acad Sci U S A 1992; 89(10):4452–6; PMID:1316610; http://dx.doi.org/10.1073/ pnas.89.10.4452 10. Jones S, Chen WD, Parmigiani G, Diehl F, Beerenwinkel N, Antal T, Traulsen A, Nowak MA, Siegel C, Velculescu VE, et al. Comparative lesion sequencing provides insights into tumor evolution. ProcNatl Acad Sci U S A 2008; 105(11):4283–8; PMID:18337506; http://dx.doi.org/10.1073/pnas.0712345105
e970069-3
Revealing human intestinal stem cell and crypt dynamics Ann-Marie Baker* and Trevor A Graham* Evolution and Cancer Laboratory; Barts Cancer Institute; Barts and the London School of Medicine and Dentistry; Queen Mary University of London; London, UK
Keywords: clonal evolution, crypt life-cycle, human intestine, neutral drift, stem cell
Stem cell and crypt dynamics in the human gut have been remarkably poorly characterized. We used random somatic mutations to trace stem cell lineages in the human intestine and coupled these data with mathematical modeling to infer the in vivo temporal dynamics of human intestinal stem cells.
The application of transgenic lineage tracing technology has led to tremendous advances in our understanding of the in vivo behavior of adult stem cells in model organisms. The stem cell biology of the intestinal crypt is a prime example. Crypts are finger-like invaginations present along the entirety of the intestinal epithelium, and the flux of cells along the crypt (production at the crypt base, migration along the crypt axis, and shedding into the lumen) implies that each crypt is sustained by the clonal expansion, vertical migration, and differentiation of basally located colonic stem cells (See Fig. 1). In mice, the application of transgenic lineage tracing has led to the identification of a set of “marker” genes (such as Lgr51) that are expressed by the basal stem cells. Subsequent mathematical modeling of clone-level data has quantified the continual competition that occurs between stem cells for the retention of a position in the niche.2-4 However, human intestinal stem cell behavior has remained poorly described because transgenic lineage tracing strategies cannot be applied to humans. In our study recently published in Cell Reports,5 we described for the first time a novel method of examining in vivo stem cell behavior within the human colon. We used naturally occurring somatic mutations to trace clonal lineages in the human intestinal crypt, coupled with mathematical modeling to infer the temporal dynamics of stem cell evolution.
To detect somatic mutations we used loss of activity of the mitochondrially-encoded enzyme cytochrome c oxidase (CCO), which can be attributed to an underlying somatic mutation.6 Since a CCO mutation that occurs in a long-lived stem cell will be inherited by all of its progeny, we were able to use CCO loss of activity (CCO¡) to track clonal populations within the colon.7 We examined serial en-face sections throughout the length of human colonic crypts in which CCO¡ clones in each section were visualized using enzyme histochemistry (with CCO¡ cells appearing blue and CCOC cells brown). We then stacked the serial sections to form a reconstruction (or ‘map’) of the crypt in question in which CCO¡ clones can be seen to arise in the crypt base and extend through the crypt in a blue ‘clonal ribbon’ (see Fig. 1). We realized that the change in width of the CCO¡ ribbon along the crypt axis is governed by the division of stem cells at the base. When a CCO¡ stem cell is lost from the niche the ribbon width decreases, and conversely when a CCO¡ stem cell divides and displaces a CCOC stem cell from the niche, the ribbon gets wider. The changes in ribbon width along the crypt axis are effectively a short-term record of events in the stem cell niche, as the distance along the vertical axis of the crypt is proportional to the elapsed time since the cells were generated at the crypt base. Thus, to infer the in vivo behavior of human intestinal stem cells, we quantified
the deviations in CCO¡ ribbon width along the crypt axis and then performed a mathematical analysis of these data, modeling the changes in ribbon width as a random walk. We found that normal human colonic crypts contain a small number of functional stem cells (approximately 5–6), a similar number to the murine crypt.8 Importantly, we showed that increases in ribbon width are balanced by decreases in ribbon width, demonstrating neutral drift of the stem cell population. In addition to analyzing healthy human crypts, we studied tissue from patients with familial adenomatous polyposis (FAP) who carry a germline mutation in one copy of the tumor suppressor gene adenomatous polyposis coli (APC).9 Loss of the remaining copy of APC leads to adenoma development. We found an increase in stem cell number and the loss/replacement rate in FAP adenomas, indicating that the parameters of stem cell competition within the niche are altered. Interestingly, although stem cell dynamics are fundamentally different within FAP adenomatous crypts, they remain governed by a neutral drift process. We recognize that our estimations of stem cell dynamics were based on historical measurements of certain parameters, and that the margin of error of these values will of course impact the accuracy of our calculations. In addition to our estimations of stem cell dynamics, we were also able to exploit CCO mutations to examine the dynamics of crypt division, the process responsible for
© Ann-Marie Baker and Trevor A Graham *Correspondence to: Ann-Marie Baker; Email: [email protected]; Trevor A Graham; Email: [email protected] Submitted: 09/02/2014; Revised: 09/04/2014; Accepted: 09/06/2014 http://dx.doi.org/10.4161/23723548.2014.970069
www.landesbioscience.com
Molecular & Cellular Oncology
e970069-1
Figure 1. Inferring intestinal stem cell dynamics from clonal imprints on the crypt wall. Schematic showing a colonic crypt with a ribbon of cytochrome c oxidase-deficient cells (CCO¡, blue color) migrating from the crypt base to the top of the crypt; this ribbon is a “clonal imprint” on the crypt wall. Changes in the CCO¡ ribbon width (termed “wiggles”) along the crypt axis are evident. We hypothesized that changes in ribbon width represent a record of the dynamics of the stem cells at the crypt base; specifically, that increases in the ribbon width were caused by replacement of a wild-type stem cell (brown) with a CCO¡ stem cell (blue), whereas decreases in the ribbon width were due to loss of a CCO¡ stem cell from the pool. By quantitatively analyzing the changes in ribbon width, we were able to infer the temporal dynamics of stem cell evolution.
early adenoma growth. When a CCO¡ stem cell expands to take over the stem cell niche, the crypt will become entirely CCO¡, and if this crypt then undergoes sequential fission events there will be a ‘patch’ of adjacent CCO¡ crypts. We analyzed the size and distribution of such patches mathematically (using a birth-process model) to determine the crypt division rate. We found that a normal colonic crypt divides once every 30–40 years, and in FAP adenomas this rate increases by an order of
magnitude. This suggests that even in adenomas the fission rate is remarkably slow, a result consistent with the observed slow progression of adenoma to carcinoma.10 In summary, we quantified the size of somatic mutant clones in the human intestine to estimate in vivo stem cell and crypt dynamics in health and disease. We found that stem cell number, stem cell loss/replacement rate, and crypt fission rate are elevated in FAP adenomas, indicating an important role for APC in the
regulation of stem cell and crypt dynamics. Our methodology of mathematically inferring clonal dynamics from static images is readily transferrable to other tissues and tumors and therefore has potential to reveal stem cell biology throughout the human body.
References
449(7165):1003–7; PMID:17934449; http://dx.doi. org/10.1038/nature06196 2. Lopez-Garcia C, Klein AM, Simons BD, Winton DJ. Intestinal stem cell replacement follows a pattern of neutral
drift. Science 2010; 330(6005):822–5; PMID:20929733; http://dx.doi.org/10.1126/science.1196236 3. Ritsma L, Ellenbroek SI, Zomer A, Snippert HJ, de Sauvage FJ, Simons BD, Clevers H, van Rheenen J. Intestinal crypt homeostasis revealed at single-stem-cell
1. Barker N, van Es JH, Kuipers J, Kujala P, van den Born M, Cozijnsen M, Haegebarth A, Korving J, Begthel H, Peters PJ, et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 2007;
e970069-2
Molecular & Cellular Oncology
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Volume 1 Issue 3
level by in vivo live imaging. Nature 2014; 507 (7492):362–5; PMID:24531760; http://dx.doi.org/ 10.1038/nature12972 4. Snippert HJ, van der Flier LG, Sato T, van Es JH, van den Born M, Kroon-Veenboer C, Barker N, Klein AM, van Rheenen J, Simons BD, et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 2010; 143 (1):134–44; PMID:20887898; http://dx.doi.org/ 10.1016/j.cell.2010.09.016 5. Baker AM, Cereser B, Melton S, Fletcher AG, Rodriguez-Justo M, Tadrous PJ, Humphries A, Elia G, McDonald SA, Wright NA, et al. Quantification of crypt and stem cell evolution in the normal and neoplastic human colon. Cell Rep 2014; 8:940–7; PMID:25127143; http://dx.doi.org/10.1016/j. celrep.2014.07.019
www.landesbioscience.com
6. Taylor RW, Barron MJ, Borthwick GM, Gospel A, Chinnery PF, Samuels DC, Taylor GA, Plusa SM, Needham SJ, Greaves LC, et al. Mitochondrial DNA mutations in human colonic crypt stem cells. J Clin Invest 2003; 112(9):1351–60; PMID:14597761; http://dx.doi.org/10.1172/JCI19435 7. Fellous TG, McDonald SA, Burkert J, Humphries A, Islam S, De-Alwis NM, Gutierrez-Gonzalez L, Tadrous PJ, Elia G, Kocher HM, et al. A methodological approach to tracing cell lineage in human epithelial tissues. Stem Cells 2009; 27(6):1410–20; PMID:19489031; http://dx.doi.org/10.1002/stem.67 8. Kozar S, Morrissey E, Nicholson AM, van der Heijden M, Zecchini HI, Kemp R, Tavare S, Vermeulen L, Winton DJ, et al. Continuous clonal labeling reveals small numbers of functional stem cells in intestinal crypts and adenomas. Cell Stem Cell 2013; 13(5):626–
Molecular & Cellular Oncology
33; PMID:24035355; http://dx.doi.org/10.1016/j. stem.2013.08.001 9. Miyoshi Y, Ando H, Nagase H, Nishisho I, Horii A, Miki Y, Mori T, Utsunomiya J, Baba S, Petersen G, et al. Germ-line mutations of the APC gene in 53 familial adenomatous polyposis patients. Proc Natl Acad Sci U S A 1992; 89(10):4452–6; PMID:1316610; http://dx.doi.org/10.1073/ pnas.89.10.4452 10. Jones S, Chen WD, Parmigiani G, Diehl F, Beerenwinkel N, Antal T, Traulsen A, Nowak MA, Siegel C, Velculescu VE, et al. Comparative lesion sequencing provides insights into tumor evolution. ProcNatl Acad Sci U S A 2008; 105(11):4283–8; PMID:18337506; http://dx.doi.org/10.1073/pnas.0712345105
e970069-3
