Analysis of the clonal architecture of the human small intestinal epithelium establishes a common stem cell for all lineages and reveals a mechanism for the fixation and spread of mutations

Article (PDF Available)inThe Journal of Pathology 217(4):489-96 · March 2009with79 Reads
DOI: 10.1002/path.2502 · Source: PubMed
Abstract
Little is known about the clonal structure or stem cell architecture of the human small intestinal crypt/villus unit, or how mutations spread and become fixed. Using mitochondrial DNA (mtDNA) mutations as a marker of clonal expansion of stem cell progeny, we aimed to provide answers to these questions. Enzyme histochemistry (for cytochrome c oxidase and succinate dehydrogenase) was performed on frozen sections of normal human duodenum. Laser-capture microdissected cells were taken from crypts/villi. The entire mitochondrial genome was amplified using a nested PCR protocol; sequencing identified mutations and immunohistochemistry demonstrated specific cell lineages. Cytochrome c oxidase-deficient small bowel crypts were observed within all sections: negative crypts contained the same clonal mutation and all differentiated epithelial lineages were present, indicating a common stem cell origin. Mixed crypts were also detected, confirming the existence of multiple stem cells. We observed crypts where Paneth cells were positive but the rest of the crypt was deficient. We have demonstrated patches of deficient crypts that shared a common mutation, suggesting that they have divided by fission. We have shown that all cells within a small intestinal crypt are derived from one common stem cell. Partially-mutated crypts revealed some novel features of Paneth cell biology, suggesting that either they are long-lived or a committed Paneth cell-specific long-lived progenitor was present. We have demonstrated that mutations are fixed in the small bowel by fission and this has important implications for adenoma development.
Journal of Pathology
J Pathol 2009; 217: 489496
Published online
20 November 2008 in Wiley InterScience
(www.interscience.wiley.com) DOI: 10.1002/path.2502
Original Paper
Analysis of the clonal architecture of the human small
intestinal epithelium establishes a common stem cell for
all lineages and reveals a mechanism for the fixation and
spread of mutations
Lydia Gutierrez-Gonzalez,
1,2#
Maesha Deheragoda,
1,3#
George Elia,
1
Simon J Leedham,
1
Arjun Shankar,
4
Charles Imber,
4
Janusz A Jankowski,
1,5
Douglass M Turnbull,
6
Marco Novelli,
3
Nicholas A Wright
1,7
and Stuart AC McDonald
1,5
*
1
Histopathology Unit, London Research Institute, Cancer Research UK, London, UK
2
Institute of Health Sciences of Aragon, Zaragoza, CIBERehd, Spain
3
Department of Histopathology, University College London Hospital, London, UK
4
Department of. Surgery, University College London Hospital, UK
5
Department of Clinical Pharmacology, University of Oxford, UK
6
Mitochondrial Research Group, School of Neurology, Neurobiology and Psychiatry, University of Newcastle upon Tyne, UK
7
Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, UK
*Correspondence to:
Stuart AC McDonald,
Histopathology Unit, Cancer
Research UK, 44 Lincoln’s Inn
Fields, London, UK.
E-mail: stuart.mcdonald@cancer.
org.uk
#
These authors contributed
equally to this work.
No conflicts of interest were
declared.
Received: 17 September 2008
Revised: 21 October 2008
Accepted: 13 November 2008
Abstract
Little is known about the clonal structure or stem cell architecture of the human small
intestinal crypt/villus unit, or how mutations spread and become fixed. Using mitochondrial
DNA (mtDNA) mutations as a marker of clonal expansion of stem cell progeny, we aimed to
provide answers to these questions. Enzyme histochemistry (for cytochrome c oxidase and
succinate dehydrogenase) was performed on frozen sections of normal human duodenum.
Laser-capture microdissected cells were taken from crypts/villi. The entire mitochondrial
genome was amplified using a nested PCR protocol; sequencing identified mutations and
immunohistochemistry demonstrated specific cell lineages. Cytochrome c oxidase-deficient
small bowel crypts were observed within all sections: negative crypts contained the same
clonal mutation and all differentiated epithelial lineages were present, indicating a common
stem cell origin. Mixed crypts were also detected, confirming the existence of multiple stem
cells. We observed crypts where Paneth cells were positive but the rest of the crypt was
deficient. We have demonstrated patches of deficient crypts that shared a common mutation,
suggesting that they have divided by fission. We have shown that a ll cells within a small
intestinal crypt are derived from one common stem cell. Partially-mutated crypts revealed
some novel features of Paneth cell biology, suggesting that either they are long-lived or a
committed Paneth cell-specific long-lived progenitor was present. We have demonstrated
that mutations are fixed in the small bowel by fission and this has important implications
for adenoma development.
Copyright
2008 Pathological Society of Great Britain and Ireland. Published by John
Wiley & Sons, Ltd.
Keywords: stem cell; small intestine; mitochondrial DNA
Introduction
Unlike the mouse, little is known about the clonal
structure of the human small bowel cryptvillus unit.
Moreover, there is no experimental confirmation of
the unitarian hypothesis, whereby a single stem cell is
responsible for the generation of all cell lineages [1].
As a consequence, there is a paucity of data describ-
ing the mechanism of spread and fixation of mutations
within the small bowel. We generally subscribe to the
mutation and selection theory of tumour development
[2] and, although small bowel tumours are rare com-
pared to large bowel tumours, there is evidence of
a similar genetic progression and mutational spectrum
[3] and therefore the understanding of the clonal archi-
tecture and the mechanisms of spread of mutations
become important. At the basis of such investigations
is the intestinal stem cell, which is likely to be the
only cell type within the crypt long-lived enough to
accrue sufficient mutations for tumour development,
although definitive evidence that such cells are the
‘cancer-initiating’ cells has yet to be demonstrated.
Copyright 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
www.pathsoc.org.uk
490 L Gutierrez-Gonzalez et al
Novelli et al [4] took advantage of the X-inactiva-
tion of a defective glucose-6-phosphate dehydrogenase
(G6PD) gene in a population of Sardinian women to
show that human small intestinal crypts were clonal,
staining either positively or negatively for G6PD.
However, such clonal evolution occurs early in devel-
opment and does not tell us anything about the dynam-
ics of clonal conversion, or of lineage relationships
in the adult, or about the inter-relationships between
crypts. On the other hand, villi are thought to be poly-
clonal, with contributions from several crypts, con-
firmed in a very rare XO/XY patient with familial
adenomatous polyposis (FAP) whose villi were a mix-
ture of Y-chromosome-positive and -negative cells,
whereas the crypts were either positive or negative and
never mixed [5]. There are, however, some important
questions that remain unanswered:
1. How are the numbers of crypts and villi in the
human small bowel maintained?
2. How do mutations spread within the small bowel
epithelium?
3. Is there an adult stem cell from which all differen-
tiated epithelial cells are derived?
4. Does niche succession and monoclonal conversion
(the processes by which a single stem cell takes
over the entire stem cell population and by which
the progeny of single stem cell displaces others,
respectively) occur in small intestinal crypts, as has
been recently shown in the colon [6] and stomach
[7]?
To answer these questions we have used mitochon-
drial DNA (mtDNA) mutations as a marker of clonal
expansion of stem cell progeny. We have previously
shown that such mutations are established in stem
cells within the human gastrointestinal tract and are
passed onto their progeny [68]. To date, this is the
only experimental means of lineage tracing within
normal adult human tissues. Mitochondria are the pre-
dominant generators of cellular adenosine triphosphate
(ATP) and within each cell are varying numbers of
mitochondria, within which are varying numbers of
mitochondrial genomes. mtDNA is a simple 16.6 kb
circular DNA that encodes 13 essential proteins of the
mitochondrial oxidative phosphorylation complexes,
22 rRNAs and 2tRNAs. We have exploited the fact that
mtDNA is susceptible to mutations due to poor repair
mechanisms, no protective histones and residing in
a highly oxidative environment [9]. Somatic mtDNA
mutations appear randomly and can only be detected
in the human intestine in patients around 40 years
of age, the number increasing exponentially with age
[6,8,10,11]. They can affect all mtDNA copies (homo-
plasmy) or a proportion (heteroplasmy), and homo-
plasmy or a high degree of heteroplasmy is required in
order for a mutated cellular phenotype to be observed.
In this article, we show that human small bowel
crypts can be entirely deficient in the mitochondrially-
encoded enzyme cytochrome c oxidase and, through
mutational analysis, reveal that every cell in such
a deficient small bowel crypt contains the same
mtDNA mutation. This shows that such crypts are
clonal. We also observe mixed crypts (containing both
cytochrome c oxidase positive and negative cells),
strong evidence that there are multiple stem cells
within the small bowel crypt. Furthermore, analysis
of some mixed crypts has revealed some interesting
features of small bowel crypt stem cell biology: the
Paneth cells are positive but every other cell type is
negative, suggesting that Paneth cells are far longer-
lived than other differentiated epithelial cells. We also
show that mutated crypts can spread by fission, a
process in which a crypt bifurcates, leading to two
independent yet related crypts, not shown before in
the human small intestine.
Materials and Methods
Patients
Ten patients undergoing a pancreaticoduodenectomy
for pancreatic tumours were used in this study (six
male, four female; age range, 4574 years). All duo-
denal specimens used were morphologically normal,
showing no sign of inflammation or dysplasia. The
specimens were either frozen, muscularis layer fac-
ing down on a glass slide in liquid nitrogen-cooled
isopentane, or formalin-fixed and paraffin-embedded.
Ethical approval was sought and obtained according
to the requirements of the UK Human Tissue Act
(2006) from the Joint UCL/UCLH Hospitals Commit-
tees on the ethics of human research (A) (Reference
04//Q0504/31).
Enzyme histochemistry
Frozen sections were cut at a thickness of 8 µm.
Sequential cytochrome c oxidase and succinate dehy-
drogenase (SDH, the presence of which was used to
highlight any deficiencies in cytochrome c oxidase)
histochemistry was performed, as previously described
[6,8]. Briefly, sections were incubated in cytochrome c
oxidase medium containing 100 m
M cytochrome c,
20 mg/ml catalase and 4 m
M diaminobenzidine tetrahy-
drochloride in 0.2
M phosphate buffer, pH 7.0, all
sourced from Sigma-Aldrich (Poole, UK), for approx-
imately 30 min, then washed in phosphate-buffered
saline (PBS) buffer, pH 7.4, for 3 × 5 min. The sec-
tions were then incubated with SDH medium (130 m
M
sodium succinate, 200 mM phenazine methosulphate,
1m
M sodium azide and 1.5 mM nitroblue tetrazolium
in 0.2
M phosphate buffer, pH 7.0) for 45 min at 37
C
or until a strong blue stain had developed, then washed
in PBS for 3 × 5 min. The sections were dehydrated
in an ethanol series, cleared in Histoclear (Lamb Lab-
oratory Supplies, Eastbourne, UK) and mounted with
Permount and allowed to dry overnight.
J Pathol 2009; 217: 489496 DOI: 10.1002/path
Copyright
2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Mutation spread in the human small bowel 491
Immunohistochemistry
Paraffin sections were cut at 2 µm thick and allowed to
air-dry, then were dewaxed in xylene and rehydrated
through decreasing alcohol concentrations. Endoge-
nous peroxidase was blocked by methanol/H
2
O
2
.The
sections were then microwaved for 10 min in boil-
ing sodium citrate buffer, pH 6.0, and allowed to
slowly cool under running distilled water. Then the
sections were incubated in a serum-free protein block
(DAKO, Ely, UK) for 10 min, followed by strepta-
vidin for 15 min and then biotin, also for 15 min, at
room temperature (Vector Laboratories, Peterborough,
UK). Primary antibodies were applied for 35 min at
room temperature in a humid chamber. The primary
antibodies used were; mouse anti-human cytochrome c
oxidase subunit 1 (Molecular Probes Invitrogen, Pais-
ley, UK; 1 : 250), Chromogranin A (DAKO; 1 : 50),
rabbit anti-human lysozyme (DAKO; 1 : 2000) and
CD10 (Abcam; 1 : 200). All antibodies were diluted
in PBS with 5% fetal calf serum. The sections were
then washed for 3 × 5 min in PBS, followed by
30 min of incubation with appropriate secondary anti-
bodies conjugated to biotin. The sections were incu-
bated with streptavidin peroxidase for another 30 min,
washed, and developed in a solution containing 4 m
M
diaminobenzidine and 0.2% hydrogen peroxide. Sec-
tions were dehydrated through graded alcohols, cleared
with xylene and mounted with DPX.
We used formalin-fixed paraffin-embedded sections
for lineage tracing rather than frozen sections because
of their better resolution with the lineage-specific
antibodies we selected. Taylor et al [8] have previ-
ously shown that immunohistochemistry, using anti-
cytochrome c oxidase (subunit 1), reliably detects
cytochrome c oxidase deficiency. Alcian blue/dias
tase/periodic acidSchiff (all reagents from Sigma,
UK) staining was performed as previously described
[7]. Briefly, sections were dewaxed and rehydrated,
then incubated in 1% Alcian blue/3% acetic acid for
5 min and washed in water. The sections were then
incubated in 1% periodic acid for 8 min, washed in
water, then covered in Schiff’s reagent for a further
8 min; they were then incubated for 15 min in diastase
at 37
C, washed and counterstained in haematoxylin.
The sections were then dehydrated through increasing
alcohol concentrations, cleared in xylene and mounted
with coverslips using DPX.
Isolation of DNA from cells
Frozen sections (20 µm thick) were cut onto UV-
irradiated PALM membrane slides (PALM Microlaser
Biotechnologies, Bernried, Germany). Cells were care-
fully cut into sterile, UV-irradiated, 0.5 ml PALM
tubes with adhesive caps, using a PALM laser msys-
tem. We attempted to laser-capture single cells; how-
ever, due to technical limitations, we were unable
to guarantee that a single cell was cut. Laser cap-
ture microdissection was performed multiple times on
each crypt and the colour boundaries of cytochrome c
oxidase-deficient and -positive cells (blue and brown,
respectively) were easily distinguished. Following
centrifugation at 7000 × g for 10 min, the cell was
lysed in 14
µl lysis buffer (Picopure, Arcturus, CA,
USA) at 55
C for 2 h and then denatured at 95
Cfor
10 min.
Mitochondrial DNA sequencing of individual small
bowel epithelial cells
From every cell laser-captured, we sequenced
the entire mitochondrial genome. The mitochondrial
genome was amplified in overlapping fragments by
using a series of M13-tailed oligonucleotide primer
pairs, as previously described [6]. PCR products were
sequenced by using BigDye version 3.1 terminator
cycle sequencing chemistries on an ABI Prism 3100
Genetic Analyzer (Applied Biosystems, Foster City,
CA, USA) and compared directly with the revised
Cambridge reference sequence (rCRS) by using
SEQUENCE ANALYSIS and SEQSCAPE software
(Applied Biosystems), as described [6].
Results
Multiple stem cells in the human small bowel crypt
In every patient we identified both entirely cytochrome
c oxidase-deficient and partially deficient crypts
(Figure 1AC). Interestingly, in Figure 1B the Paneth
cell zone is positive but the rest of the crypt is defi-
cient, strongly suggesting that Paneth cells are longer-
lived than the other differentiated cell types within the
small intestinal crypt. We cannot discount the possibil-
ity that other cells are also present in the Paneth cell
zone; however, we are confident that there were no
mutated Paneth cells in this crypt. As expected, villus
epithelium can appear mixed if surrounding crypts are
either positive or deficient in cytochrome c oxidase,
demonstrating that multiple crypts feed the epithelium
of a single villus. The presence of partially-mutated
crypts demonstrates the presence of multiple stem cells
within a small bowel crypt (Figure 1C), where there
at least two populations of stem cells, one cytochrome
c oxidase positive and one deficient. The presence
of multiple stem cell clones suggest that partially-
mutated crypts are in a state of flux where the crypt
will eventually become entirely mutated or revert to its
wild-type state niche succession and monoclonal
conversion. Interestingly, we also observed patches of
mutated crypts (Figure 1D) indicating that these crypts
had divided. Overall the percentage of mutated crypts
seen in small bowel sections was relatively low (see
Supporting information, Table S1) when compared to
those seen in the colon [6,8] but small bowel resections
are rare and it is difficult to acquire enough samples
to perform statistical analysis.
J Pathol 2009; 217: 489496 DOI: 10.1002/path
Copyright
2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
492 L Gutierrez-Gonzalez et al
Figure 1. Human small bowel crypts are clonal and contain multiple stem cells. (A) An entirely cytochrome c oxidase-deficient
crypt feeding onto a polyclonal villus. (B) A mixed crypt. The Paneth cell zone is entirely positive for cytochrome c oxidase (arrow)
but the remainder of the crypt is deficient, suggesting that there are multiple stem cells and that Paneth cells are longer-lived than
other differentiated epithelial cells. (C) A mixed crypt in transverse section (arrow). (D) A patch of cytochrome c oxidase-deficient
crypts, suggesting that a cytochrome c oxidase-deficient crypt has divided
Small bowel crypts are clonal
To demonstrate that every cell within a mutated crypt
is derived from a single stem cell clone, we laser-
captured microdissected individual cells all the way
up from the base of the crypt into the villus and
sequenced the entire mtDNA genome from each cell.
Figure 2A and B show the pre- and post-captured
crypt/villus, respectively. Figure 2C demonstrates that
each mutated (blue) cell (cell numbers 2 and 3)
contains the same mtDNA mutation throughout the
crypt (T C transition at position 879 of the MT-
RNR1 12s RNA site), which is not seen in any of
the neighbouring cytochrome c oxidase-positive crypt
cells (cell number 1) of the villus the mutated crypt
adjoins. This is strong evidence to suggest that human
small bowel crypts are clonal.
It is not possible from the dual histochemical stain-
ing to identify each of the differentiated epithelial cell
lineages within a mutated crypt, a necessary step to
show that each lineage originates from one common
stem cell. To this end we have used immunohistochem-
istry for subunit 1 of the cytochrome c oxidase gene
to identify mutated crypts in paraffin sections, then in
serial sections to demonstrate that each of the individ-
ual epithelial lineages are present within it. In Figure 3
we have shown that a cytochrome c oxidase-deficient
crypt (Figure 3A) contains goblet cells (Figure 3B,
demonstrated by Alcian blue/PAS/diastase staining),
CD10-positive enterocytes (Figure 3c), lysozyme
+
Paneth cells (Figure 3D) and chromogranin A-positive
neuroendocrine cells (Figure 3E): thus, all lineages
are mutated in this crypt and this therefore confirms
the unitarian hypothesis in the human small intesti-
nal epithelium. Figure 3F shows an isotype-matched
negative control.
Mutations are spread through the human small
bowel by crypt fission
In Figure 1D we have shown that cytochrome c
oxidase-deficient crypts can be observed in patches.
This may due to a random accumulation of cytochrome
c deficient crypts or, as is seen in the colon, indicative
that crypts themselves had divided [6]. To test this lat-
ter hypothesis we sequenced the entire mitochondrial
genome from laser-capture cells from each mutated
crypt within a patch. The sequencing was repeated
twice and multiple cells sequenced. Figure 4AC
(pre- and post-laser capture sections) shows that every
cell captured from a mutated crypt within a patch
contained an identical mutation (in this case a sin-
gle adenosine base deletion in the 66926698 polyA
region (underlined) in the MT-CO1 gene leading to a
downstream GluAsn change; arrow, Figure 4D). All
the neighbouring cytochrome c oxidase-positive crypts
showed a wild-type genotype. This is strong evidence
for the presence of the same mutated stem cell in adja-
cent crypts, which must have divided by fission in
order for a patch to develop. The odds of an identical
mutation occurring randomly in neighbouring crypts
has been previously calculated to be 1 : 2.48 × 10
9
[6]
and therefore chance is highly unlikely to account for
this.
J Pathol 2009; 217: 489496 DOI: 10.1002/path
Copyright
2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Mutation spread in the human small bowel 493
Figure 2. Each cell within a cytochrome c oxidase-deficient crypt contains the same mtDNA mutation. (A) Pre-laser capture
and (B) post-laser capture photographs of the same cytochrome c oxidase-deficient crypt. (C) Sequence analysis. Each blue cell
contains a T C transition at position 879 of the MT-RNR1 12s RNA site (cell numbers 2 and 3 shown), whereas all brown cells
are wild-type (cell 1 shown). Multiple cells (12 15 cells/section) were cut from the cytochrome c oxidase-deficient crypt and its
neighbours in serial sections with the same result. This is strong evidence that human small bowel crypts are clonal
Discussion
This article provides definitive evidence for the clonal
architecture of human small intestinal crypts and
villi. Furthermore, we present evidence that the small
bowel crypt contains multiple stem cells, yet all the
differentiated epithelial lineages within a crypt are
derived from one common stem cell. Interestingly,
our data suggest that Paneth cells are either long-lived
or that their committed progenitor is long-lived. We
have also demonstrated the mechanism, crypt fission,
by which mutations are fixed and spread within the
small bowel.
We propose that a single stem cell towards the
base of the crypt [12] accrues, over a long period of
time, sufficient mutated mitochondria that result in the
stem cell becoming cytochrome c oxidase-deficient.
The progeny produced by this stem cell will also
be cytochrome c oxidase-deficient and all lineages
are present within such mutant clones (monoclonal
conversion). We believe that this stem cell is then able
to seed the entire stem cell population of such crypts
(niche succession). If such somatic mtDNA mutations
confer a selective advantage or disadvantage [13,14]
over non-mutated stem cells it is probably mild in
the small bowelprimarily because mutated crypts are
very rare in patients under the age of 40 and even in
elderly patients are not particularly common. Work is
currently under way to study this. Moreover, intestinal
epithelial cell mutations which do confer a selective
advantage tend to reveal themselves at an earlier age,
as seen in patients with FAP who have a mutation
in Apc, and develop multiple polyps at a young age
[15]. On the other hand, if these mutations confer a
disadvantage we would expect not to see deficient
crypts, as the wild-type stem cells would dominate:
modelling of this system is required to determine this.
If there is no selective advantage, then a single stem
cell can only populate the entire stem cell zone by a
stochastic mechanism and can be lost at any time. If
it is successful, the entire crypt will become mutated.
Although we do not have sufficient samples for
statistical calculations, we have shown (see Supporting
information, Table S1), however, that there is a trend
for an increase in the number of mutated crypts
and patch size with age, although the percentage of
cytochrome coxidase-deficient crypts is low. We
observe fewer cytochrome c oxidase-deficient crypts
J Pathol 2009; 217: 489496 DOI: 10.1002/path
Copyright
2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
494 L Gutierrez-Gonzalez et al
Figure 3. All differentiated epithelial cells are derived from a single stem cell. Serial sections showing (A) an entirely cytochrome
c oxidase-negative crypt (arrow) contains: (B) PAS/Alcian blue/diastase-stained goblet cells; (C) CD10-positive enterocytes;
(D) lysozyme-positive Paneth cells; and (D) chromogranin A-positive neuroendocrine cells. (E) An isotype-matched negative
control. All intestinal epithelial cell lineages are present within a cytochrome c oxidase-deficient crypt
in the small intestine than we do in the large bowel.
The reasons for this are unclear; however, some studies
have suggested that there are more stem cells within
the small intestinal crypt [16] or that these stem cells
have a longer cell cycle time [17]. Furthermore, the
clonal stabilization time in the small intestine may
be longer than that of the large intestine, possibly
because of the stochastic process occurring in a greater
number of stem cells. However, in a recent study
(where lgr5 was proposed to be a marker of stem
cells in the murine intestine [18]) the number of
crypts arising from lgr5-positive stem cells in the small
intestine was greater than that of the large bowel.
More analysis is required to understand why there
are fewer cytochrome c oxidase-deficient crypts in the
human small bowel.
In Figure 1B we showed a crypt that was deficient
for cytochrome c oxidase in all epithelial cell types bar
the Paneth cell zone. Although we cannot discount the
possibility that there are other cells in the Paneth cell
zone [18], it could therefore be suggested that Paneth
cell progenitors are long-lived if progenitors for all the
other epithelial cell types have been replaced, at an
earlier time point, by cytochrome c oxidase-deficient
cells. To date, there have been no experimental studies
to determine the turnover time of the Paneth cell
population in the human small bowel crypt. In mice
it is estimated to be around 23 days [19], which is
a long time in comparison to enterocytes, which are
completely replaced within 72 h. If we extrapolate
from the lifespan of a mouse (approximately 2 years)
to that of a human (approximately 70 years), then
an estimated turnover time of human Paneth cells
would be 2.2 years. We appreciate that this is a very
rough estimate indeed and is likely to be inaccurate;
however, the presence of cytochrome c oxidase-
positive Paneth cells in an otherwise cytochrome c
oxidase-deficient crypt would lend credence to our
hypothesis that Paneth cells are the longest-lived
differentiated cells within a crypt. We believe that
in the example shown in Figure 1B the crypt has
already undergone niche succession with cytochrome
J Pathol 2009; 217: 489496 DOI: 10.1002/path
Copyright
2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Mutation spread in the human small bowel 495
Figure 4. Human small bowel crypts divide by fission. (A) A patch of cytochrome c oxidase-deficient crypts pre-laser capture and
(B, C) post-laser capture. (D) Sequence analysis. Every cytochrome c oxidase-deficient cell cut from every deficient crypt within
a patch contained the same mutation. In this case an adenosine base deletion in the 66926698 polyA region (underlined and
highlighted with
)intheMT-CO1 gene leading to a downstream GluAsn change (highlighted by the arrow) was observed in each
deficient crypt (cell numbers 2 and 3 shown). This was not present in neighbouring wild-type crypts (cell 1 shown). Therefore, a
founder crypt has divided by fission to form a patch of related crypts
c oxidase-deficient stem cells (including Paneth cell
progenitors) but, since fully differentiated Paneth cells
are long-lived, cytochrome c oxidase-positive Paneth
cells persist.
To conclude, we have shown here that human small
bowel crypts are clonal, with all the differentiated lin-
eages originating from a common stem cell, whereas
the contribution of several crypts leads to villi being
polyclonal. We have demonstrated the presence of
multiple stem cells, and that one stem cell can populate
the entire stem cell zone whose progeny will populate
the entire crypt, so-called niche succession and mon-
oclonal conversion. We have shown that patches of
mutated crypts share the same mtDNA mutation and
therefore one parent mutated crypt must have divided
by fission, over time, to produce daughter crypts shar-
ing the same mutation. We would hypothesize that this
is the mechanism by which mutations spread in the
human small bowel. This work has also revealed inter-
esting insights into the longevity of Paneth cells and
provides us with the tools to calculate the Paneth cell
turnover time; but many more specimens are required
for this.
Acknowledgements
SACM and JAZ are funded by Oxford University and Cancer
Research UK. LG is funded by the Institute of Health Sciences
of Aragon, Spain.
J Pathol 2009; 217: 489496 DOI: 10.1002/path
Copyright
2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
496 L Gutierrez-Gonzalez et al
Supporting information
Supporting information may be found in the online
version of this article.
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J Pathol 2009; 217: 489496 DOI: 10.1002/path
Copyright
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    • "Infrequent stochastic loss of CCO activity (CCOÀ) is observed in the human intestine and is attributed to an underlying somatic mitochondrial DNA (mtDNA) mutation (Taylor et al., 2003). mtDNA sequencing confirms that adjacent CCOÀ cells in the intestine are clonally derived (Fellous et al., 2009; Greaves et al., 2006; Gutierrez-Gonzalez et al., 2009; Taylor et al., 2003). CCO activity was assessed in en face serial sections of colonic mucosa (n = 9 patients;Table S1). "
    [Show abstract] [Hide abstract] ABSTRACT: Human intestinal stem cell and crypt dynamics remain poorly characterized because transgenic lineage-tracing methods are impractical in humans. Here, we have circumvented this problem by quantitatively using somatic mtDNA mutations to trace clonal lineages. By analyzing clonal imprints on the walls of colonic crypts, we show that human intestinal stem cells conform to one-dimensional neutral drift dynamics with a ‘‘functional’’ stem cell number of five to six in both normal patients and individuals with familial adenomatous polyposis (germline APC�/+). Furthermore, we show that, in adenomatous crypts (APC�/�), there is a proportionate increase in both functional stem cell number and the loss/replacement rate. Finally, by analyzing fields of mtDNA mutant crypts, we show that a normal colon crypt divides around once every 30–40 years, and the division rate is increased in adenomas by at least an order of magnitude. These data provide in vivo quantification of human intestinal stem cell and crypt dynamics.
    Full-text · Article · Aug 2014
    • "Since only stem cells have long enough life spans to accumulate mtDNA mutations to detectable levels of homoplasmy, a clonal population marked by a particular mtDNA mutation likely represents the progeny of a single mutated stem cell (Elson et al., 2001; Greaves et al., 2006;). Using this lineage tracing methodology, stem cell niches have been located in many human tissues (Elson et al., 2001; McDonald et al., 2008; Gutierrez-Gonzalez et al., 2009; Lin et al., 2010; Gaisa et al., 2011b). Our use, for the first time, of whole-mount human lung imaging allowed us to use quantitative analysis of these clones to demonstrate both that basal cells are the likely multipotent progenitor cells in the human upper airways and that airway homeostasis is maintained in a stochastic manner. "
    [Show abstract] [Hide abstract] ABSTRACT: Lineage tracing approaches have provided new insights into the cellular mechanisms that support tissue homeostasis in mice. However, the relevance of these discoveries to human epithelial homeostasis and its alterations in disease is unknown. By developing a novel quantitative approach for the analysis of somatic mitochondrial mutations that are accumulated over time, we demonstrate that the human upper airway epithelium is maintained by an equipotent basal progenitor cell population, in which the chance loss of cells due to lineage commitment is perfectly compensated by the duplication of neighbours, leading to “neutral drift” of the clone population. Further, we show that this process is accelerated in the airways of smokers, leading to intensified clonal consolidation and providing a background for tumorigenesis. This study provides a benchmark to show how somatic mutations provide quantitative information on homeostatic growth in human tissues, and a platform to explore factors leading to dysregulation and disease. DOI: http://dx.doi.org/10.7554/eLife.00966.001
    Full-text · Article · Oct 2013
    • "As new crypts arise from rapid expansion of resident stem cells and subsequent fission, these polyclonal crypts become monoclonal. This clonality is maintained throughout adulthood, and can serve to fix genetic and epigenetic changes geographically within the intestines (Fuller et al., 1990; Endo et al., 1995; Novelli et al., 1996) For example, mutations can arise in crypts and these mutant crypts can expand by fission to create patches of clonally-derived cells with identical mutations (Greaves et al., 2006; Gutierrez-Gonzalez et al., 2009). Epigenetic marks are similarly stable and can be used to study stem cell dynamics in the intestines (Yatabe et al., 2001). "
    [Show abstract] [Hide abstract] ABSTRACT: The endoderm gives rise to the lining of the esophagus, stomach and intestines, as well as associated organs. To generate a functional intestine, a series of highly orchestrated developmental processes must occur. In this review, we attempt to cover major events during intestinal development from gastrulation to birth, including endoderm formation, gut tube growth and patterning, intestinal morphogenesis, epithelial reorganization, villus emergence, as well as proliferation and cytodifferentiation. Our discussion includes morphological and anatomical changes during intestinal development as well as molecular mechanisms regulating these processes.
    Full-text · Article · Mar 2011
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