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Whole population cell kinetics of mouse duodenal jejunal iieal and colonic epithelia as determined by radioautography and flow cytometry.

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THE ANATOMICAL RECORD 203:251-264 (1982)
Whole Population Cel1 Kinetics of Mouse Duodenal,
Jejunal, Ileal, and Colonic Epithelia as
Determined by Radioautography and
Flow Cytometry
HAZEL CHENG A N U MATTHEW BJERKNES
Department of Anatomy, Medical Sciences Building, University of Toronto,
Toronto, Ontario, Canada M5S IA8
ABSTRACT
We report methods for the determination of whole population
cell kinetics in the mouse intestinal epithelium by means of radioautography and
flow cytometry. Epithelium was isolated from the four regions of the mouse intestine by the perfusion method described in Bjerknes and Cheng (1981a).Two experimental series were performed. In the first series, the tissue from one set of animals was fixed in 3.5% paraformaldehyde, dissociated by serial filtration, and
then processed for flow cytometry. In the second series, fixed epithelium from a
second set of animals was dissociated by gentle pipetting and then used to prepare
dried cell suspensions on slides which were radioautographed. The whole population kinetic parameters determined by the two techniques, flow cytometry and
radioautography, were not significantly different, indicating the reliability of both
techniques for whole population kinetics determinations. In al1 regions of the intestinal tract, 12-14% of epithelial cells were in the S-phase. From this value, the
whole epithelial turnover time was calculated to be about 60 hours in al1 regions of
the intestine. The whole epithelial growth fraction was calculated to be 0.23 for the
small intestinal epithelium and 0.31 for the colonic epithelium. Detailed analysis
of the flow cytometric data showed that there were significantly more cells in early
S-phase than in mid and late S-phase. From the isolated epithelial sheets a mean
number of crypts per villus was determined for the duodenum, jejunum, and
ileum. Single villi and crypts were microdissected for the preparation of squashes.
From the squashes a mean number of cells per villus as wel1 as per crypt was determined. From the results, the ratio of the number of epithelial cells in the villus population to the number of cells in the crypt population was determined to be 1.41,
1.31, 1.36 in duodenum, jejunum, and ileum, respectively. The proportion of the
three main epithelial cell types, columnar, mucous, and Paneth, was determined
with the dried cell suspension preparations. There was a decreasing gradient in the
proportion of columnar cells from the proximal to the distal intestine while an
increasing gradient was observed in the proportion of mucous cells. We found that
mucous cell divisions account for only one half of mucous cell production in the
small intestine. In the colonic epithelium, mucous cell divisions account for twothirds of mucous cell production. This was in agreement with previous findings.
The intestinal epithelium has a complex
three-dimensional arrangement. This complexity has made the study of whole epithelial kinetics difficult. This is reflected in the range of
values reported in the few attempts at such
measurements in the rat (Leblondand Stevens,
1948; Bertalanffy,1960; Altmann and Enesco.
1967).
The difficulty in making accurate whole pop-
0003-276X18212032-0251$04.00
1982 ALAN R. LISS. INC
ulation measurements is primarily technological. Conventionalmethods do not allow accurate
measurement of a tissue with the topological
complexity of the intestinal epithelium. For ex~
-
~
~
~
Keceived Fehrudry 20. 1981 accepted December 14. 1981
Matthew Hjerknes's present address is Department of Anatomy.
University of Minnesota. 4~135Jackson Hall. 321 Church Street S.E..
Minneapolis. Minnesota 55455.
252
H. CHENG AND M. BJERKNES
ample, if whole population kinetic measurements are made on tissue sections, many complex errors wil1 be introduced. What is needed
is a method that allows the study of the epithelium as a whole, and that is free of selection
artifacts. In this paper we introduce one such
approach.
Recently, we reported methods for the isolation of structurally intact epithelium, free of
underlying nonepithelial elements, from any
segment of mouse intestine (Bjerknes and
Cheng, 1981a).The integrity of the preparation
insures the true representation of al1 epithelial
elements. This makes it suitable for whole population kinetic measurements. In this paper we
report the application of this preparation to
the study of whole population intestinal epithelial cell kinetics using radioautography and
flow cytometry. These methods allow US to
make accurate whole population measurements
of epithelial labeling and mitotic indices. In addition, methods are als0 presented for differential counting of the main epithelial cell types
with resulting whole population measurements
of their behavior.
MATERIALS AND METHODS
Animals
Al1 animals were adult male Swiss albino
mice (25-35 gm). They were housed in animal
facilities, maintained on a light-dark cycle of
7:OO to 19:OO hours light, and were given free
access to Purina laboratory chow and water.
Isolation procedure for intestinal epithelium
The isolation procedure has been reported
previously in detail (Bjerknes and Cheng,
1981a). Briefly, animals were given an intraperitoneal injection of 1 &i of 3H-thymidine
per gm body weight 1 hour before they were
killed (al1 animals were killed between 11:OO
A.M. and noon). They were anesthetized with an
intraperitoneal injection of 60 mglkg sodium
pentobarbital and were killed by intraventricular perfusion with a 37" C solution of 30 mM
ethylenediamine tetraacetic acid (EDTA) in
calcium-magnesium-free Hank's balanced salt
solution (CMF).The duration of perfusion was
3 minutes. At the end of the perfusion, a 5-cm
segment of duodenum (immediately distal to
the pylorus), jejunum (16 cm distal to the pylorus), ileum (immediately proximal to the
ileocecal valve), and colon (proximal to the
rectum) were collected and placed in cold CMF.
The segments were everted and placed on glass
rods. The rods were mounted in a stirrer-vibrator (Buchler Scientific) and the gut was
vibrated in tubes of cold CMF. The vibration
released large sheets (up to 2 cm2)of pure epithelium. Al1 of the epithelium from each segment was isolated.
Fixation
The epithelium was allowed to settle on ice.
The CMF was aspirated and replaced with 30
cc of a cold 3.5% solution of paraformaldehyde
in CMF (pH 7.4). The samples were spun for 4
minutes at 500 g in a refrigerated centrifuge.
Then the fixative was aspirated and the samples were washed four times with cold CMF.
The samples were then stored in about 5 cc of
CMF at 4" C at least overnight before use.
Preparation of slides for radioautography
An aliquot of the epithelium, about 0.5 ml
(0.5 X 106 cells), was removed, pelleted, the
CMF aspirated, and the epithelium resuspended in about 0.25 ml of distilled water. Then
the epithelium was gently pipetted with a
pasteur pipette to disperse the cells. After
about five strokes, a smal1 drop of the sample
was examined with phase-contrast microscopy. Usually, at this point most of the sample
was dissociated. If the cells were not mostly
dispersed, the pipetting could be prolonged
until dispersion was complete. (The sample
was checked routinely to ensure that no broken
cells were foiind. If broken cells were observed
the sample was discarded and the dissociation
process repeated with a new aliquot.) A drop or
two of the sample was placed on a 37" C slide
warming tray to dry. Therefore the preparation used was a dried cell suspension. The
average time for the procedure, from dissociation to preparation of slides, was about 3 minutes. The slides were then dipped in methanol.
After they were dried they were prestained
with alcian blue for 30 minutes then processed
for radioautography (Kodak NTB-2 emulsion;
2 weeks exposure; Kodak D-19 development;
Kopriwa and Leblond, 1962). The slides were
poststained through the emulsion with
Harris's hematoxylin for 30 minutes, xanthene
for 1 minute, and then mounted for study.
Labeling and mitotic index counts
The radioautographs were examined at a
magnification of X 1,000. The fields scored
were 1 mm apart from each other as measured
by the stage micrometer. Cells with ten or
more silver grains over their nuclei were con-
INTESTINAL EPITHELIAL CELL KINETICS
sidered labeled. Every cell in the field was
scored as being either labeled or not. Mitotic
figures were als0 recorded. Three slides per animal were used and about 1,000 cells were
scored from each slide. Therefore a total of
about 3,000 cells were scored per animal. From
the results, the labeling and mitotic indices
were determined for the whole epithelium. A
total of 13 animals was studied for duodenum,
19 for jejunum, 16 for ileum, and 14 for colon.
Differential counts of the epithelial cells
Another series of animals were used for differential cell type count determination. The
radioautographs were analyzed in the manner
described above. Every cell in the field was
scored; its cell type and whether or not it was
labeled was recorded. The percentage of each
cell type and its labeling index (when applicable) was calculated for each animal and the
mean for al1 the animals determined. A total of
seven animals were studied for duodenum, six
for jejunum, nine for ileum, and nine for colon.
Preparation of cells for flow cytometry
Another series of animals were used for flow
cytometry. A total of five animals were studied
for duodenum, eight for jejunum, eight for
ileum, and seven for colon.
The cells were dispersed by passage through
a series of nylon meshes (20 pm Nitex). Each
sample was first passed five times through one
layer of Nitex in a 13-mmsweeney filter holder
(Millipore Inc.), then three times through two
layers of Nitex, three times through three
layers, and finally five times through four
layers. The filters were routinely checked after
each step for residual clumps of cells, and if
clumps were found, the sample was passed
through that filter three additional times. This
was usually sufficient to disperse the clumps.
If not, the sample was discarded and another
aliquot was used. After dispersion, a drop of
each sample was examined for doublets with
phase-contrast microscopy. Samples with
more than 1% doublets were discarded and
another aliquot was used. For this method, the
sample volume should be kept to about 1 cc
(1-2 x i 0 6 cells). If a Iarger sample volume is
necessary, the sample should first be dispersed
by gentle pipetting and then passed through
the series of filters. The filtration is necessary
after the pipetting to break up smal1 clumps
and doublets; without it. doublets are as high
as 3%.
After dispersion, the cells were pelleted and
253
resuspended in a solution of 0.1Y0 RNAase
(Sigma type IA) in Dulbecco’s phosphate-buffered saline (PBS).They were incubated at 37”
C for 2 hours. Then the cells were pelleted and
resuspended in a solution of 0.025% propidium
iodide (Sigma)in 0.037 M sodium citrate. After
5 minutes staining in the dark, the cells were
pelleted and resuspended in PBS. The cells
were routinely examined with ultraviolet
microscopy and no background cytoplasmic
staining was observed. The samples were then
kept in the dark until analysis with the flow
cytometer.
Flow cytometry
The samples were first filtered five times
through four layers of Nitex and then run in
our TPS-1 cell sorter (Coulter Electronics Inc.).
Our sorter has a 30 mW argon ion laser at 488
nm. The sheath fluid was PBS. The sample flow
rate was kept under 300 cellslsecond. For each
animal, about 20 runs (a total of 100.000 to
200,000 cells) were analyzed. The data were
transferred int0 an interfaced Z-80-based microcomputer (Arisia Microsystems Inc., Mississauga, Ontario, Canada) running under the
CPlM operating system (Digital Research Inc.,
California). Each run from each animal was
normalized to a population of 100,000 cells. A
mean DNA histogram for each animal was
then calculated from the normalized runs.
Analysis of the D N A histograms
For the duodenum and colon the mean DNA
histogram from each animal was analyzed independently by the method of Fried and Mandel (1979)using a copy of their multigaussian
curve-fitting routine. The program was run on
the DEC-10 computer at the University of
Toronto Computing Center. The results were
als0 analyzed on our microcomputer system
using the multigaussian curve-fitting routine
described in the Appendix. In addition to the
kinetic parameters obtained with the Fried
program, the program described in the Appendix provides conclusions about the distribution of cells in four different subcompartments
within the S-phase (see Appendix). Since we
found no significant differences in kinetic parameters as determined by the Fried program
and those determined on our microcomputer
(see Results), data for the jejunum and ileum
were analyzed by the microcomputer-based
program alone.
We als0 derived an “average”epithelial histogram for each region of the intestine by taking
254
H. CHENG AND M. BJEHKNES
the average of the mem DNA histograms from
each of the animals used.
Direct determination of the crypt to uillus ratio
Five animais were used in this study. The
epithelium from a segment of duodenum, jejunum, and ileum was isolated directly int0
2.5% glutaraldehyde for maximum preservation of the structural integrity. The isolated
epithelium was fixed for 30 minutes in the
Same fixative. An epithelial sheet was chosen
from each animal and the total number of villi
and crypts in the sheet was counted under a
stereoscope. From these results the number of
crypts per villus was determined for each
region of the small intestine. Another epithelial sheet from each animal was stained for 7
minutes with Harris hematoxylin. Villi and
crypts were microdissected (great care was
taken to leave the surface epithelium with the
villi). Four villi and four crypts were randomly
selected from each animal, dehydrated with
graded ethanol, and mounted individually with
permount on microscope slides. The villi or
crypts were then slowly squashed to disperse
the epithelial cells. The number of cells per
villus or crypt was determined from these
slides. The ratio of villus cells to crypt cells was
calculated for each region of the small intestine
by multiplying the number of crypts per villus
by the number of cells per crypt and then
dividing the result int0 the number of cells per
villus.
Statis tical methods
Results from different animals were kept distinct. Means and associated error values were
determined for any given parameter by considering the mean of the results from each animal
as independent samples, determining sums
and sums of squares, and then using as N the
number of animals studied. Means were compared with “Student’s’’t-test. The probability
level used for a decision of a significant difference was P s 0.05.
RESULTS
A typical example of a radioautograph of a
dried cell suspension is shown in Figure 1.
Three of the four main epithelial cell types
(columnar, mucous, and Paneth) were easily
identified (Fig. 2). Enteroendocrine cells were
not clearly defined in these preparations. We
have not developed methods to clearly demonstrate them. However, in terms of the whole
population kinetic parameters measured here,
the inability to distinguish this population
from the columnar cells was not significant
(enteroendocrine cells represent less than 1%
of the epithelial population and have essentially the Same turnover characteristics as do
the columnar and mucous cells; Cheng and
Leblond, 1974a,b).
Whole population kinetic parameters of
mouse intestinal epithelium as derived from
the radioautographs are shown in Table 1. The
Fig. 1. Photomicrograph of an average radioautograph of a dried cell suspension. X 550.
255
INTESTINAL EPITHELIAL CELL KINETICS
Fig. 2. Photomicrographs of b i e d cell suspension preparation showing in a) a mucous cell, b) a Paneth cell (arrow-
TABLE I . Whole population kinetic parameters
head), and c) a Paneth cell (arrowhead)and columnar cells. X
900.
of mouse intestinal epithelium determined by radioautography
S-phase ‘70
(X i S.E.)
G2-phase%
(X i S.E.)
M-phase %
(X I S E . )
G ,-phase ‘70
(X I S.E.)
Duodenum
13.0 f 0.52
1.0 f 0 . 2 4
83.2 I 1.11
Jejunum
12.3 f 0.72
0.8 I 0.05
84.1 f 0.73
Ileum
13.3 f 0.43
0.8 f 0 . 0 5
83.0 f 0.61
Colon
12.2 i 0.52
3.0
2.6
2.5
2.6
2.5
2.7
2.5
2.5
0.8 i 0.03
84.3 f 0.67
f 0.72’
f 0.10’
f 0.16’
I 0.12’
f0.15‘
IO.10’
f 0.10’
f0.11’
‘Value for G2-phase calculated from the M-phase a s follows:
M.I. X
Gi-phase duration
M-phase duration =
E
0.5
2Value for Gi-phase calculated frorn the S-phase a s follows:
% S-phase
Gi-phase duration
= 9% S-phase X E .
7.4
S-phase duration
proportion of cells in G2-phasewas calculated
from both the S-phase and M-phase as shown
in the footnotes to Table 1. The proportion of
cells in the G1-phase was then determined.
There were no significant differences in the kinetic parameters of al1 the regions of the intestinal tract studied.
With our procedure for flow cytometry, the
G peak of the DNA histograms routinely had
a coefficient of variance (CV) in the 4-570
range. The kinetic parameters derived from
analysis of the duodenal and colonic data with
Frieds program were not significantly different (Table 2) from those derived with the micro-
computer-based program (see Appendix).
Therefore the jejunal and ileal data were only
analyzed with the microcomputer-based program. As in the case of the radioautographic
study there were no significant differences in
the kinetic parameters of the four regions of
the intestine. There were no significant differences between the results derived with flow cytometry and those derived with radioautography for any of the four regions. Table 3
shows the distribution of cells in four subcompartments within the S-phase as determined
by the microcomputer-based program. In al1
four regions of the intestine there was a signifi-
256
H. CHENG AND M. BJERKNES
TABLE 2. Whole population kinetic parameters of mouse intestinal epithelium determined by f i w cytometry
G2 + M-phase 90
(X f SE.) - _ ~ _ _
S-phase %
(X f S.E.)
Duodenum
13.1
12.4
12.7
13.6
13.2
12.6
Jejunum
Ileum
Colon
4.1
3.9
3.6
3.6
4.5
4.2
f 0.94’
f 1.43’
I 1.02’
f 1.01’
f 0.78’
f 0.85’
G ‘-phase %
(X f .S.E.)
- ~-
f0.23’
f 0.34’
f 0.38’
i 0.24’
f 0.50’
f 0.51’
82.9
83.8
83.8
82.8
82.4
83.2
f 0.95’
f 1.20’
f 1.35’
f 0.91’
i 1.19’
f 1.32’
’ Analyzed hy the Fried and Mande1 program.
’Andyzed by the microcomputer-haced program.
TABLE 3. Uistribution of epithelial cells in the four subcompartrnents within the S-phase
Duodenum
Jejunum
Ileum
Colon
Subcompartment 1
Subcompartment 2
Subcompartment 3
70
90
%
(X I S.E.)
(X f S.E.)
(X f S.E.)
20.5 1 0 . 9 2
19.1 I
2.33
13.4 I
1.12
17.1 I 1.50
23.3 f 2.16
18.0 f 1.67
15.1 f 1.84
23.9 k 1.18
37.6
40.6
54.6
40.3
f 5.14
f 5.05
f 5.10
I 2.94
cantly larger proportion of cells in the first subcompartment than in the remaining three subcompartments of S-phase. Figures 3-10 show
plots of the “average”DNA histograms determined from the mem DNA histograms studied
for duodenum, jejunum, ileum, and colon.
The proportion of the three main cell types,
columnar, mucous, and Paneth, in the whole
epithelial population is shown in Table 4. The
proportion of labeled cells within each cell
types is ais0 included in the table. The proportion of columnar cells was the Same in duodenum and jejunum but was significantly lower
in ileum and colon. There was, however, no difference in the proportion of labeled cells in the
columnar cell populations throughout the intestinai tract. The proportion of mucous cells
was the same in duodenum and jejunum but
was significantly higher in the ileum and even
higher in the colon. However, no significant
differences were found between regions in the
proportion of labeled cells in the mucous cell
population. The percentage of Paneth cells
was significantly higher in ileum than in the
duodenum and jejunum.
In the duodenal epithelium there were large
variations in the size of both villi and crypts.
Some villi were about two to three times as
wide as the average duodenal villus while some
crypts were iiiuch longer than the average duo-
4
20
40
Subcompartment 4
90
(if S.E.) ~
18.7
22.3
16.9
18.8
GO
8[!
f 2.24
i 2.07
f 2.31
f 2.01
i I!?
i .2
’
CHRhlUFl NclVRFFi
Fig. 3. DNA histogram of mouse duodenai epithelium.
The data presented are the average derived from the means
of five animals and are fitted by the microcomputer-based
prugraixi.
~
257
INTESTINAL EPITHELIAL CELL KINETICS
2500
u7
J
J
2000
LL
o
U
D
cr
1 SU0
W
u7
z
3
z
i000
500
__L.li
20
GO
80
i00
i20
CHFlhNEL hdMBER
Fig. 4. The data presented in Figure 3 were plotted on a
different scale to show details of the S-phase and G * + M
phase curves.
Fig. 5. DNA histogram of mouse jejunal epithelium. The
data presented are the average derived from the means of
eight animals and are fitted by the microcomputer-based
program.
i50~ci
2500
m
i
2
Cu
i)
2003
LL
O
I
!
1
,
i 508
i 00ii
100
CYcihhFI
[U,JPQrCi
Fig. 6 The data presented in Figure 5 were plotted on a
different scale to show details of the S-phase and Gz+M
phase curves.
Fig. 7. DNA histogram. of mouse ileal epithelium. The
data presented are the average derived from the means of
eight animals and are fitted by the microcomputer-based
program.
denal crypts. This did not appear to be the case
in jejunum and ileum. Both the viUi and crypts
in those locations appeared to be of relatively
constant size. These observations are reflected
in the variation of the duodenal measurements
shown in Table 5.
The cel1 count studies for crypts and villi
confirmed previous reports that the size of
H. CHENG AND M. BJERKNES
2500
2000
1500
1000
500
20
40
GO
80
i00
1 2 ~
CHQNNCL VdWSF?
Fig. 8. The data presented in Figure 7 were plotted on a
different scale to show details of the S-phase and Gz+M
phase curves.
200UU
s,
i
15OCO
Fig. 10. The data presented in Figure 9 were plotted on a
different scale to show details of the S-phase and G,+M
phase curves.
both villi and crypts decreases from the proximal to the distal region of the small intestine.
Such a decreasing gradient was als0 observed
in the number of crypts associated with a villus. The ratio of villus cells to crypt ceìls, however, was similar throughout the length of the
small intestine. (Table 5 ) .
Lu
o
DISCUSSION
LL
D
5000
Fig. 9. DNA histogram of mouse colonic epithelium. The
data presented are the average derived kom the means of
seven animals and are fitted by the microcomputer-based
program.
Accurate whole population epitheiial kinetic
parameters are significant. Following perturbation (physiologicalor otherwise),a response observed in the crypt population may be reflected
very differently at the level of the whole population. For example, if there was an increase in
the size of the nonproliferative compartment
(largely made up of the villus compartment)
while the proliferative compartment remained
constant, whole population measurements
would detect the change while crypt measurements probably would not. Conversely, if there
was a decrease in the proliferative population
and a proportional decrease in the size of the
nonproliferating population, no detectable
change would be seen at the whole population
level. However, at the crypt level, significant
changes would be observed. So, while crypt
measurements are important, whole population measurements are als0 necessary for a
259
INTESTINAL EPITHELIAL CELL KINETICS
T A B L E 4. Proportion of the three main cell tyDes in mouse intestinal epithelium
Columnar
Labeling index
(Ef S.E.)
(X f S.E.)
Whole population
labeling index
(i f S.E.)
Duodenum
Jejunum
Ileum
Colon
12.6
11.9
12.4
12.2
f 0.64
i 0.96
f 0.62
f 0.52
%
97.0
96.6
93.4
84.2
I 0.22
f 0.48
I 0.66*
I 0.713
12.9 I 0.68
12.2 f 1.03
12.9 f 0.66
13.1 I 0.56
90
(Xf S.E.)
Mucous
Labeling index
(X I S.E.)
2.5 i 0.13
2.9 f 0.49
5.5 I 0.53*
15.8 f 0.713
5.1
5.1
6.1
6.9
Paneth
70
(Xf S.E.)
f 0.99
f 0.85
f0.89
10.52
0.5 f 0.12
0.6 10.18
1.1 i 0.20'
O
'Significantly different from the duodenal population iP < 0.05).
'Significantly different from the duodenal and jejunal populations (P < 0.011.
'Significantly different from the duodenal. jejunal. and ileal populations (P i0.01)
T A B L E 5. EDithelial cell counts of intestinal uilli and crvuts
# cells per villus
# cells per crypt
# crypts per villus
a
(i I S.E.)
b
(X f S.E.)
C
(X f S.E.)
881 f 93.7
620 i 47.7'
P < 0.05
403 f 47.g2
P < 0.02
10.2 i 0.43
8.5 i 0.34'
P < 0.02
6.9 f 0.312
P < 0.01
~~~~~~~~
Duodenum
Jejunum
Ileum
~~~
~~~~~~~~
12.638 f 2.369
6:918 I '8073
P < 0.02
3.775 f 3162
P < 0.02
~
~~
ratio of villus cells
to crypt cells
aibc
~~~
~
1.41
1.31
1.36
'Significantly different from duodenum and ileum.
2Significantly different from duodenum and jejunum
'Significantly different from ileum.
true picture of the overall response of the intestinal epithelium.
This paper is an attempt to derive accurate
whole population epithelial kinetic parameters
using isolated epithelium from various regions
of the intestinal tract of normal adult mice.
The parameters reported were derived with
two different techniques, radioautography and
flow cytometry. There were no significant differences between the two sets of data (Tables
1, 2). The correspondence of the two sets of
data demonstrates that both techniques,
radioautography and flow cytometry, can be
used to derive reliable whole population kinetic
parameters of intestinal epithelium. While
flow cytometry is a more rapid procedure,
radioautography does not require specialized
equipment, which may not be available to
some laboratories.
Pipetting was used for the dissociation of
cells in preparation for radioautography while
the epithelium was dissociated by filtration for
flow cytometry. The similarity of the vahes
derived from radioautography and flow cytometry indicates that we were not selectively
losing cells during the filtration-dissociation
step in flow cytometry.
Whole population kinetic parameters
Kinetic parameters for the crypt population
determined by radioautographic studies on tissue sections of mouse intestine give a labeling
index of about 33yo for the crypt population in
duodenum, 30% in jejunum, and 29% in ileum
(derived from the data reported in Cheng and
Leblond, 1974a).The ratio of villus cell number
to crypt cell number was determined to be
about 1.41 for duodenum, 1.31 for jejunum,
and 1.36 for ileum (Table 5). Therefore the labeiing index for the whole epithelium can be estimated to be 13.7% (3311.41 1)for duodenum, 13% (3012.31) for jejunum, and 12.3%
(29/2.36)for ileum. These numbers correspond
wel1 with the direct measurements of labeiing
index made in the present study (Tables 1, 2)
and further indicate the reliability of the whole
population kinetic parameters reported in this
paper.
As shown in Tables 1and 2 there were no significant differences in the kinetic parameters
+
260
H. CHENG AND M. BJERKNES
of the four regions studied. At first glance, this
homogeneity in kinetic parameters might be
surprising. I t is known that there is a clear reduction in villus size from the proximal to the
distal portion of the small intestine to the extreme of no villus compartment in the colon
(Altmann and Leblond, 1970). Intuitively,
such an obvious decrement in the overall size
of this substantial nonproliferating compartment should be reflected in regional variation
in whole epithelial kinetic parameters. However, the reader must realize that there is a corresponding reduction ;n the size of the crypts
and in the number of crypts per villus (Table 5;
Fujii, 1972; Wright, 1980). Thus there is a simultaneous reduction in the size of both the
proliferating and the nonproliferating compartments. As a result the ratios of villus cells
to crypt cells were similar throughout the
small intestine (Table 5; Wright, 1980).The kinetic parameters at the whole epithelial level
were therefore not biased by variation in the
size of the nonproliferating villus compartment. In the case of colon, one might expect a
higher labeling index at the whole epithelial
level than that in the small intestine because
of the absence of the nonproliferating villus
compartment. However, in the colon there is
the surface epithelium, a nonproliferating compartment of significant size. Furthermore, the
cell cycle time for the proliferating cells in colon is longer than that in the small intestine
(Appleton et al., 1980). These factors were
probably responsible for the value of the whole
population labeling index observed in the colonic epithelium. When al1 of these factors are
taken int0 account, our finding of a homogeneity in whole population kinetic parameters
throughout the intestinal tract appears
reasonable.
Relatiue sizes of the crypt and uilluspopulations
The ratio of the number of epithelial cells in
the villus population to the number of cells in
the crypt population was found to be similar
along the length of the small intestinal epithelium (Table 5 ) . This finding is in agreement
with the report of Wright (1980). The villusi
crypt cell ratio can als0 be estimated algebraically from the labeling indices of the crypt
population and of the whole epithelium (WLI,
whole population labeling index; CLI, crypt labeling index; NLC, number of labeled cells;
NVP, number of cells in villus population;
NCP, number of cells in crypt population;
using the equations WLI = NLCI(NCP
NVP), and CLI = NLCINCP and then solving
+
for NVPINCP we derive the equation NVPI
NCP = (CLI - WL1)IWLI).Using a crypt labeling index of about 3370, 30%. and 29% for
duodenum, jejunum, and ileum, respectively,
and the whole population labeling indices determined in this study (Table l),the ratio of villus cells to crypt cells was calculated to be 1.5,
1.4, and 1.2 for duodenum, jejunum, and ileum,
respectively. These estimates correspond wel1
with the direct measurements made in this
study (Table 5). Since the two sets of numbers
were determined by two independent methods
(one from the labeling indices, the other from
actual cell counts) their similarity again
indicates the reliability of the whole population kinetic parameters reported in this
paper.
Whole epithelial turnover time
With conventional methods it is difficult to
accurately determine the turnover time for the
whole epithelium. Using the labeling indices
determined in this paper (Tables 1, 2 ) , and a
mean S-phase duration of 7.4 hours (ts)for intestinal epithelial cells (Schultze et al., 1972;
Kovacs and Potten, 1973; Appleton et al.,
1980), the turnover time (100 X t,ILI) for the
whole epithelial cell population can be calculated. The results are displayed in Table 6. Al1
four regions had a whole epithelial turnover
time of about 60 hours.
By definition, turnover time is the time it
takes to produce a number of cells equal to the
total number of cells present in the population
(Cheng and Leblond, 1974a).Therefore, whole
epithelial turnover time determinations should
include al1 cell types. Al1 cells were included in
the determination of the 60-hour turnover time
discussed above. However, the specific turnover characteristics of each of the cell types
may vary. For example, the Paneth cellpopulation has a turnover time of about 3 weeks
(Bjerknes and Cheng, 1981b) while the columnar cells have a turnover time of about 58
hours (Table 6).
Whole epithelial growth fraction
The whole epithelial growth fraction may
als0 be approximated from our results by the
equation GF = LII(100 X t4tc).With a small
intestinal cell cycle time (tc)of about 13 hours
(Al-Dewachiet al., 1975; Schultze et al., 1979),
and a whole population labeling index (LI) of
about 13%(Tables 1 , 2 ) ,thegrowth fraction for
the small intestinal epithelium can be estimated to be about 0.23. In other words, in the
small intestinal epithelium, about 23% of the
26 1
INTESTINAL EPITHELIAL CELL KINETICS
TABLE 6. Whole population, columnar cell. and mucous cell turnouer characteristics
Duodenum
(k t S.E.)
Whole population
turnover time'
Columnar cell
turnover time'
Mucous cell
turnover time'.2
Jejunum
_ _ (X f S.E.)
Ileum
-
(i f S.E.)
-.
59.7 k 3.01
63.6 f 4.30
60.9 I 2.94
62.0 ? 2.73
58.2 f 3.02
62.1 f 4.34
58.4 I 2.85
58.0 t 2.55
181.3 -+ 26.3
191.5 f 45.1
143.5 I 21.5
124.0 I 16.4
'Turnover time for each population is calculated as follows:
x 100 where tS is the duration of S-phase 17.4 hoursi and L.I. is the labeling index of the cel1 population
'This is not the true mucous cell turnover time because columnar cells contribute heavily to the mucus cell h e .
cells are proliferating. The growth fraction in
the colon is higher. With a mean tc of about
18.5 hours (Appletonet al., 1980)and a labeling
index of about 12.5% (Tables 1, 2), the colonic
growth fraction is 0.31. Thus in the colon,
about 31% of al1 epithelial cells are
proliferating.
Mucous cell renewal
I t has been demonstrated that in the crypt,
both the columnar and the mucous cell populations turn over at about the same rate (Merzel
and Leblond, 1969; Cheng, 1974; Cheng and
Leblond, 1974b; Chang and Nadler, 1975).
Cheng (1974)as well as Chang and Nadler (1975)
further demonstrated that in the crypt, the
rate of proliferation of mucous cells is too low
to account totally for mucous cell renewal. I t
was concluded that the stem cell and thus the
columnar cell population contribute to the renewal of the mucous cell population (Cheng,
1974; Cheng and Leblond, 1974b).Our present
data confirm these previous findings.
If we assume that the mucous cell population receives no external contribution to its
numbers, then using the columnar cell S-phase
duration (according to Thrasher and Greulich,
1966, mucous and columnar cells have the
same S-phase duration), and the labeling index
of mucous cells (Table 4), the turnover time of
the mucous cell population could be calculated
(this value is not the true mucous cell population turnover time because, as is discussed below, columnar cells contribute heavily to the
mucous cell line).These values were included in
Table 6. This calculated turnover time was
much longer than the turnover time of the columnar cell population. Since the mucous cell
population is actually renewing at about the
same rate as the columnar cell population we
confirmed our previous finding (Cheng, 1974;
Cheng and Leblond, 1974b) that the mucous
cell population did not produce cells at a rate
that will explain their rate of renewal. In order
for the columnar and mucous cell populations
to be renewed at about the same rate, cells
other than mucous cells must contribute to the
renewal of the mucous cell population. If the
turnover time of the columnar cell line is calculated (Table 6), it may be seen that they are renewing at a slightly higher rate than the whole
population (the difference is not significant;
however, since the mucous cell population is
much smaller than the columnar cell population it will only need a very smal1portion of the
columnar cells to meet the need of the mucous
cell renewal rate). Thus we confirm that the columnar cell population is participating in mucous cell renewal.
I t is possible to calculate the proportion of
the mucous cell population derived from mucous cell divisions (as opposed to those derived
from columnar cells). Following the rationale
outlined in Cheng (1974), given that the
S-phase duration and rate of renewal are similar for both columnar and mucous cells, if the
mucous cell population alone was responsible
for its renewal then the frequency of cells in
either line (columnar or mucous) should be proportional to their corresponding fraction within the proliferating compartment of the epithelium. For example, the percentage of mucous
cells in the epithelium should be proportional
to the percentage of the labeled epithelial cells
that contain mucus (i.e.,if there are 100 labeled
epithelial cells and ten of them contain mucus,
then the percentage of the labeled epithelial
cells that contain mucus will be 10% and this
should be equal to the percentage of mucous
cells in the epithelium). Similarly, the labeling
262
H. CHENG AND M. BJERKNES
indices of the two cell lines (columnar and mucous) should be the same. On the other hand, if
the columnar cell line were contributing to the
mucous cell population, then the percentage of
mucous cells would be higher than the percentage of labeled epithelial cells with mucus, and
the labeling index of the mucous cell population would be lower than that of the columnar
cell line. The latter case was observed (Tables
4,7).The ratios (labelingindex of mucous cellsi
labeling index of columnar cells, percentage
labeled cells with mucuslpercentage epithelial
cells with mucus) should indicate the relative
contribution of mucous cell divisions to the
mucous cell population. The remainder would
come from the columnar cell line. These number are shown in Table 7. Thus, only about half
of the small intestinal mucous cell line is derived from mucous cell divisions. This is in
agreement with our previous determination for
the crypt mucous cell population using conventional radioautographic studies on tissue sections (Cheng, 1974). In the colonic epithelium
approximately two-thirds of the mucous cells
were derived from mucous cell divisions (Table
7). Thus, the mucous cell line in the colon is
more actively proliferating than that in the
small intestine. This is in agreement with the
observation of Chang and Nadler (1975)for the
colonic crypt mucous cell population using
conventional radioautographic studies on tissue sections. These results again demonstrate
the agreement of the findings derived from
kinetic parameters of the crypt population
(determined by conventional radioautographic
studies on tissue sections) with those derived
from kinetic parameters of the whole epithelium (determined by flow cytometry and radioautography on dried cell suspension). Therefore we are confident that the whole population
kinetic parameters, and thus, the techniques
reported in this paper are reliable.
In conclusion, we would like to stress the potential utility of the techniques presented in
this paper for the determination of whole epithelial kinetic parameters. They should be useful for rapid assessment of the effects of an experimental or physiological manipulation on
cell proliferation in the intestinal epithelium.
APPENDIX
Multigaussian curve fitting (such as the algorithms developed by Fried and his collegues;
Fried, 1976;Fried and Mandel, 1979)has proved
to be a sensitive method for the analysis of DNA
histograms from flow cytometric studies.
However, the usual methods for deriving these
fits require the solution of large sets of simultaneous linear equations. Because of this,
these programs require the use of large computer systems. This has meant that most users
could not have analyses computed “on the
spot” but rather had to transmit the data to a
large mainframe computer and then get the results printed at some remote location. To get
around this difficulty some effort has been expended in the development of algorithms that
would allow the analysis to be performed locally on a microcomputer. The most successful
of these has been the polynomial fitting routines of Dean and collegues (Dean and Jett,
1974; Dean, 1980). However, we have found
that the polynomial fitting algorithms do not
provide satisfactory results with our tissue.
We report here a microcomputer-compatible
algorithm we have developed which allows the
fitting of multigaussian curves to flow cytometric data. The program fits as many curves
as there are channels between the G, and the
G2 peaks. The program runs on our system in
TABLE 7. PercentaEe of mucous cell population deriued from mucous cell diuisions
% labeled cells with mucus
1 hour after 3H-thymidine
% epithelial cells with
mucus
% of mucous cell population
contributed by proliferative
mucous cells
Duodenum
(X f S.E.)
(X I S E . )
1.1 i 0.16
1.2 f 0.27
2.9 i 0.55
10.4 f 1.12’
2.5 f 0.13
2.9 f 0.49
5.5 f 0.53
15.8 f 0.71
45.6 I 6.523
44.7 i 6.68‘
42.3 f 6.453
41.4 I 6 . 4 4 ‘
53.0 f 7.923
52.1 f 7.454
‘Significantly different P < 0.01) from duodenum. jejunum. and ileum
’Significantly different ( P < 0.05) from duodenum and jejunum.
2ca,culated
as Yo labeled cells with mucus
x 100.
YOepithelial ceik with mucous
4C.culated as labeling index of mucous cells
x 100.
labeling inaex of columnar cells
Jejunum
Ileum
Colon
(X I_
S E ._
)
- ~
_ _ (X
_i S.E.)
’
63.6 i 4.46’.3
60.2 I 4.554
INTESTINAL EPITHELIAL CELL KINETICS
about 90 seconds on the average (our system is
composed of the 8-bit microprocessor made by
Zilog Corp., the Z-80A CPU, 64K bytes of
RAM and an Intel C8231A arithmetic processor chip, which doubles the speed of most arithmetic computations). The program is written
in PLlI (Digital Research Corp.) and requires
about 50K bytes to run. The algorithm is a twopart fitting procedure. First, an initial estimate of the population is made using the quadratic polynomial algorithm of Dean (1980).The
coefficient of variance is estimated by using
the method outlined by Fried and Mandel
(1979). This initial estimate is then transformed into a multigaussian approximation by
using the result obtained from the polynomial
fit for each channel as the area that must be
found under the gaussian curve that will represent the data of that channel (each of the
curves is assumed to have the same coefficient
of variance). After this approximation is
obtained, the program proceeds to the second
step. In the second step, the program searches
for the point of maximum deviation of the data
from the fit. The area under the gaussian curve
whose mean is at the channel of maximum
deviation is then adjusted in a direction that
reduces the chi square of the fit. After that
channel has been adjusted to the optimum
point, the program proceeds until each channel
has been adjusted. Then the program starts
over again, looping back through the second
stage of the program until adjustments no
longer make significant changes in the fit. In
most cases it requires four loops to fit the data.
At this stage the fitting is completed and all
that remains is the calculation of the S-phase
subcompartments. With a coefficient of variance of 4-5%, we are not justified in dividing
the S-phase into more than about four subcompartments. For this reason, the Gaussian
curves that represent the S-phase component
of the cell cycle are placed into four groups. For
example, if the G , peak was centered on channel 41, and the GZ+ M peak was centered on
channel 82, then the 40 curves that represented S-phase cells would be subdivided into
four groups. Thus, the proportion of cells in the
first subcompartment of S-phase would be calculated from the area under the Gaussian
curves centered on channels 42-51, the next
compartment would correspond to the curves
of channels 52-61, and so forth. This would
result in S-phase being broken down into four
steps, corresponding to early, early-mid, latemid, and late S-phase. These subcompartments are comparable and may be used to
263
make comparisons of the relative proportion of
cells in early, mid, or late S-phase.
ACKNOWLEDGMENTS
We thank R. Kuk for technical assistance.
Supported by the Canadian Foundation for Ileitis and Colitis and the Medical Research
Council of Canada.
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