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Transplantation of Hematopoietic Stem Cells Obtained by a Combined
Dye Method Fractionation of Murine Bone Marrow
By Ian McAlister, Norman S. Wolf, Maria E. Pietrzyk, Peter S. Rabinovitch, Gregory Priestley, and Brian Jaeger
Hematopoietic stem cells were purified from murine bone
marrow cells (BMC). Their characteristic density, size,
internal complexity, Hoechst 33342 dye uptake, and wheat
germ agglutinin (WGA) affinity were used to distinguish
them from other cells in the bone marrow. BMC suspensions were centrifuged over Ficoll Lymphocyte Separation
Media (Organon Teknika, Durham, NC; density 1.077 t o
1.08). The lower-density cells were drawn off, stained with
Hoechst and labeled with biotinylated WGA bound t o
streptavidin conjugated t o phycoerythrin (WGA-B*A-PE)
or with WGA conjugated t o Texas Red. These cells were
then analyzed and sorted by an Ortho Cytofluorograph
50-H cell sorter. The cells exhibiting medium t o high
forward light scatter, low t o medium right angle light
scatter, low Hoechst intensity, and high WGA affinity were
selected. Sorted BMC (SBMC) were stained with Romanowsky-type stains for morphologic assay, and were
assayed in lethally irradiated (LI) mice for their ability to
produce colony-forming units in the spleen (CFU-SI and for
their ability to produce survival. The spleen seeding factor
for day 8 CFU-S upon retransplantation of the isolated cells
was 0.1. The isolated cells were found to have consistent
morphology, were enriched up to 135-fold as indicated by
day 8 CFU-S assay, 195-fold as indicated by day 14 CFU-S
assay, and 150 sorter-selected BMC were able t o produce
long-term survival in LI mice with retention of donor karyotype. When recipients of this first transplantation were
themselves used as BMC donors, their number of day 8 and
day 12 CFU-S were found t o be reduced. However, 3 x 10’
of their BMC provided 100% survival among secondary
recipients. When the previously SBMC were competed
after one transplantation against fresh nonsorted BMC in a
mixed donor transplant, they showed the decline in hematopoietic potency normally seen in previously transplanted
BMC. We conclude that the use of combinations of vital
dyes for fluorescence-activated cell sorting (FACS) selection of survival-promoting murine hematopoietic stem cells
provides results comparable with those produced by antibody-selected FACS and has the advantage of a method
directly transferable to human BMC.
0 1990 by The American Society of Hematology.
H
cures for selected diseases and in bone marrow replacement
therapy.
Gradient sedimentation and cell sorting can purify stem
cells from whole bone marrow suspensions. Visser et all’ used
density gradient sedimentation, light scatter properties, and
wheat germ agglutinin (WGA)-affinity of the stem cells to
achieve high enrichment of CFU-S. Pallavicini et all2 used
light scatter properties and the low Hoechst 33342 dye
uptake characteristics of the stem cell to achieve 100-fold
enrichment of day 8 and day 12 CFU-S [CFU-S(8) and
CFU-S( 12)]. By combining these different methods of distinguishing different stem cell properties, we have demonstrated
a reliable and effective means of isolating hematopoietic stem
cells with a single passage through the fluorescence-activated
cell sorter (FACS). These cells were morphologically consistent, produced spleen colonies with a spleen seeding factor
(f-factor) of 0.1, and as few as 150 to 300 sorted cells
provided long-term hematopoiesis and survival for LI primary and secondary recipient mice.
EMATOPOIETIC STEM cells are the source of all
hematopoiesis. Through the process of differentiation
they produce the many different cell types found in the bone
marrow and peripheral blood. Several subpopulations of
hematopoietic stem cells exist, beginning with a pluripotent
stem cell capable of producing all hematopoietic lineages and
ending with lineage-restricted stem cells.’-6 Subpopulations
of early hematopoietic stem cells have been identified by
their ability to produce colony-forming units in the spleen
(CFU-S) or in vitro clonal growth and by reconstitution of
the hematopoietic system of lethally irradiated (LI) recipient
mice receiving fractionated donor bone marrow cells (BMC)
and providing for their ~ u r v i v a l . ~ ~ ’ ~
Purification of hematopoietic stem cells is an important
step toward being better able to characterize the functional,
morphologic, and membrane surface properties of stem cells,
as well as their gene activity. Purified preparations of donor
cells capable of reconstituting the hematopoietic system of
LI mice are sought for purposes of basic research into the
processes of differentiation and stem cell self-renewal. Purified stem cells may also play a role in molecular engineering
From the Department of Pathology, University of Washington,
Seattle, WA.
Submitted July 19. 1989; accepted November 22.1989.
Supported by Grant No. AGO1 751from the National Institute on
Aging.
Address reprint requests to Norman S. Wolf. DVM, PhD.
Department of Pathology SM-30, University of Washington, Seattle, WA 981 95.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
o 1990 by The American Society of Hematology.
0006-4971/90/7506-0010$3.OO/0
1240
MATERIALS AND METHODS
Animals. Three- to six-month-old (C57BL/6 x DBA)FI, henceforth referred to as BDFl, mice were used. In most experiments,
male donor cells were injected into female recipients. The parental
pure lines, derived from the NIA reference strains and the hybrids
were bred and raised in our facilities under strict specific-pathogenfree barrier conditions. They were free of all testable viral, mycoplasmal, and bacterial mouse pathogens. We tested for the following
viruses at 3-month intervals: Sendai, mouse hepatitis virus, pneumonia virus of mice, Reovirus-3, murine encephalomyelitus virus, and
minute virus of mice. In addition, a recent outside test (February
1989) by Charles River Professional Services for the above viruses
plus lymphocytic choriomeningitis, ectromelia, papova K virus,
polyoma, mouse adenovirus, epizootic diarrhea of infant mice,
murine cytomegalovirus, mouse thymic virus, and Hantaan virus
found all to be negative. Routine tests for Salmonella species,
Staphylococcus aureus. Klebsiella pneumoniae. Corynebacterium
Blood, Vol 75, No 6 (March 15). 1990: pp 1240-1246
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TWO-DYE SORTING AND TRANSPLANTATION OF HSC
kutsceri, Pseudomonas aerughosa, Yersinia pseudo TB, Y enterocolitica. Pasturella multocida. P pneumotropica, Bordetalla bronchisepta, Epierythrozoon cunijormis and endo- and ectoparasites
were also negative. Sacrifices were by cervical dislocation.
Cell suspension and density gradient fractionation. BMC were
flushed from femurs and tibias of male donor BDFl with Hank’s
Balanced Salt Solution (HBSS). The suspension was drawn through
decreasing bore needles and passed through a 100 mesh per inch
stainless steel Swinney filter (Millipore, Bedford, MA ) to produce a
single cell suspension. The cells were then layered on Lymphocyte
Separation Media (LSM, density 1.077 to 1.080 at 2OOC; Organon
Teknika, Durham, NC) and fractionated by density gradient centrifugation (500g for 15 minutes at 2OOC). The low-density lymphocytic
band was aspirated by pipette, washed three times with HBSS, and
subjected to the following procedure.
Hoechst staining and WGA labeling. The density fractionated
cells were resuspended in HBSS containing 10 pmol/L Hoechst, 20
mmol/L HEPES, 1 g/L d-glucose, and 10% fetal bovine serum
(FBS) at a final pH of 7.2. This suspension was incubated for 1 hour
at 37OC and then placed on ice. From this point on, the cells were
maintained at 4OC to preserve the intracellular Hoechst content. The
cells were washed once before being resuspended in 1 pg/mL
biotinylated WGA (WGA-B) in phosphate-buffered saline (PBS)
and incubated for 20 minutes on ice. The cells were then spun down
again and quickly resuspended in 5 pg/mL streptavidin conjugated
with phycoerythrin (PE) in PBS. In some experiments, Texas Red
(TR)-labeled WGA was substituted for the biotinylated form, and
the remainder of the format remained the same.
Cell sorting. Dual laser multivariate analysis and cell sorting
was performed at 4OC under sterile conditions by an Ortho Cytofluorograph 50HH (Ortho Diagnostic Systems, Westwood, MA). Monochromatic 35 1- to 364-nm light was used for Hoechst dye excitation
and forward light scatter measurements. Monochromatic 488-nm
light was used for PE excitation and right angle light scatter. For TR
excitation, 568-nm light from a dye laser was used. Filters used to
collect fluorescence emission were 420 nm (long pass) for Hoechst,
575 nm (band width 30 nm) for PE, and 600 nm (long pass) for TR.
The blast region of the scattergram containing 10% of the total
population was selected and analyzed for Hoechst intensity. Three
percent of these cells were selected for their low Hoechst intensity.
The TR or PE intensity of the low Hoechst cells from the blast cell
region was then analyzed, and 60% of these cells were selected as
WGA positive, the combined selection was accomplished in a single
passage of the cells through the sorter. These cells were collected in
test tubes coated with 10% FBS, 0.2 mol/L N-acetyl-d-glucosamine,
and HBSS, then diluted to the appropriate cellularity in 0.2 mol/L
N-acetyl-d-glucosamine in HBSS for removal of the WGA complex
before injection.
CFU-S assay and survival study. Young adult BDFl mice were
lethally irradiated with 11.0 Gy by a dual 13’Cssource (Gammacell40) delivering 1.36 Gy/min. This radiation dose is uniformly lethal
within 15 days in radiation control mice under our conditions, and no
endogenous spleen colonies are formed. Normal control BMC or
fractionared BMC were injected via lateral tail vein into separate
groups of irradiated mice. The number of fractionated cells injected
per mouse ranged from 150 to 400 sorted BMC (SBMC). Some
animals were killed by cervical dislocation 8 or 14 days postirradiation to examine their spleens for colony formation, while others were
allowed long-term survival. Spleens were immersed briefly ( 5 to 10
minutes) in Bouin’s solution, then placed in 10%buffered formalin,
and later observed under a dissecting scope. The f-factor was
determined for the sorted cells using the modified procedure of Till
and McC~lloch’~
as described by Wolf14 with a 3-hour primary
seeding period. Animals set aside for survival study were followed for
2 to 15 months.
1241
Morphology, staining, and analysis. Slides were washed with
absolute methanol, dried, and coated with 1% albumin. Two hundred
cells were sorted directly onto each slide. The cells were fixed with
1.5% gluteraldehyde in Cacodylate buffer by adding one drop of 2x
fixative solution directly to the drop of PBS containing SBMC.
These preparations were stained with Wright-Giemsa.
Karyotyping. Examination of BMC content for male versus
female sex chromosomes followed velban injection by 90 minutes
and was carried out by a standard method previously reported.15
Statistics. All numerical values shown in the tables are mean f
SEM where appropriate. Comparisons were made by standard
Students two-tailed t test.
RESULTS
Higher levels of purification of both CFU-S(8) and CFUS(14) were achieved with each selective step (Table 1). The
LSM procedure that selected the uppermost low-density
layer of cells increased the proportion of CFU-S(8) present
in the cell suspension fourfold, primarily removing erythrocytes and granulocytes before sorting. However, this step
routinely reduced the total number of cells by 10- to 20-fold,
so it is apparent that the majority of CFU-S(8) were lost in
this procedure. Examination of the CFU-S(8) present in the
collected meniscus versus those present in the pellet after
centrifugation indicated that only 40% of these cells were in
the fraction collected for use. Whether this reduction affected the quality of the final product is unknown. The
Hoechst exclusion step increased the proportion of CFUS(8) up to 100-fold in some experiments, and CFU-S(14) up
to 132-fold. The WGA-affinity method, when added, increased the proportion of CFU-S(8) up to 135-fold, and
CFU-S(14) up to 195-fold. The methods were additive,
providing reliable levels of high purification, as seen in the
individual experiments in Table 1.
Multivariate analysis of the light scatter, Hoechst intensity, and fluorescently labeled WGA intensity by the cell
sorter produced the following graphs. Ten percent of the
right angle versus forward light scattergram constituting the
blast region was selected (Fig 1). The cells in this region of
the scattergram were then analyzed for Hoechst fluorescent
intensity (Fig 2). Three percent of the low-intensity Hoechst
cells were selected from the Hoechst intensity histogram to
be analyzed for TR or PE fluorescence (Fig 3). The cells
positive for red fluorescence (constituting 60% of the histogram) due to their WGA affinity were then selected to be
sorted into a media-coated test tube. The cells finally selected
for CFU-S assays were of low density, blast cell light scatter
characteristics, Hoechst uptake resistant, and strongly WGA
positive. Recovery in the final collected fraction was 0.01% to
0.02% of the initially collected BMC in the typical experiment. In a typical case (Table 1, Experiment 2), Hoechst
sorting alone enriched the CFU-S(8) content 51-fold, and
the WGA method provided 5 1-fold enrichment of CFU-S(8)
independently. In the same experiment, combining the methods produced 81-fold enrichment and 24.5% purity of CFUS(8), as determined by using an f-factor of 0.1. In another
experiment (Table 1, Experiment 3), the Hoechst method
provided 53-fold enrichment of CFU-S(8) alone, and in
combination with WGA, produced 133-fold enrichment of
CFU-S(8) and 40% purity. Enrichment of CFU-S(14) was
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1242
McALlSTER ET AL
Table 1. Comparison of Hoechst and WGA Methods Independently and in Combination
~~
Group and
Day of Count
Recipient
MiceIGroup
Cells/
Recipient
Mean
CFU-SIRecipient
8
13
50,000
14.9 f 0.72
(L) 8.0 + 0.39
(TI 29.8 f 1.88
1
1
1
8
11
300
12.1 f 1.23
(L) 6.3 f 0.62
(TI 30.0 f 2.63
135.3
130.2
167.8
NBMC$
Day 8
Day 14
8
9
50,000
Hoechst only
Day 8
Day 14
7
9
200
WGA-TR only
Day 8
Day 14
6
11
402
8
9
192
10
250
4.0 f 2.5
53
16.0
9
220
8.8 f 3.0
133
40.0
Experiment 4
Hoechst only (pH 7.2)
Day 8
Day 14
8
6
300
Hoechst only (PH 7.5)
Day 8
Day 14
9
5
300
Enrichment.
Purity
(% CFU-S)t
Experiment 1
NBMCS
Day 8
Day 14
+
Hoechst
WGA-TR
Day 8
Day 14
0.3
0.2
0.6
40.0
20.8
100.0
Experiment 2
Experiment 3
Hoechst only
Day 8
Hoechst
Day 8
1
1
1
0.3
0.2
0.3
3.1 k 0.59
(L) 5.8 k 0.64
(T) 17.1 f 1.98
51.3
154.9
131.9
15.5
28.9
85.5
6.2 f 0.60
51.1
76.4
86.4
15.4
14.3
56.0
81.1
158.3
195.3
24.5
29.5
126.6
(L) 5.7 f 0.52
(TI 22.5 f 2.06
+
Hoechst
WGA-TR
Day 8
Day 14
15.1 f 1.23
(L) 9.3 f 0.44
(TI 17.1 k 3.43
4.7 f 0.75
(L) 5.7 f 3.43
(TI 24.3 f 1.29
+ WGA (WGA-E*A-PE)
5.1 f 1.3
(L) 5.3 f 0.61
(TI 31.3 f 6.58
56.9
59.2
159.5
17.0
17.7
104.3
3.1 f 0.55
34.7
73.3
229.3
10.3
22.0
150.0
(L) 6.6 i 0.92
(T) 45.0 f 5.10
Abbreviations: L, large colppies at day 14: T, total colonies at day 14.
*Enrichment = [donor NBMC/SBMC] x [CFU-~lSBMC)/CFU-S(NBMC)]. N8MC controls from Experiments 1 and 2 were used to calculate
enrichment and purification in Experiments 3 and 4.
tpurification (% CFU-S) was calculated using an experimentally derived 3 hour f-factor of 0.10 for sorted cells for CFU-S(8) (see Table 2). Thus,
multiplicationof the number of colonies formed by 10 and comparison with the number of cells injected provided the % CFU-S In the preparation.
$Sorted NBMC were not density gradient separated or sorted. All sorted cell populations were obtained from NBMC run on LSM density gradient
before sorting.
several times greater than that for CFU-S(8) (Table 1,
Experiments 1, 2, and 4), bringing the calculated degree of
purification above 100% in some cases. However, since the
small (less than 2 mm) colonies present on day 14 probably
represent descendants of secondary, ie, daughter CFU-S,’
these figures should be viewed with caution, although obviously indicating that highly self-replicative stem cells have
been selected. The larger day 14 colonies almost surely
represent continuation of day 12 colonies. Enrichment was
calculated on the basis of the number of injected cells that
were required to provide one day 8, day 12, or day 14 spleen
colony, comparing sorted preparations with untreated donor
BMC.
Spleen f-factor. The f-factors for purified CFU-S and
unfractionated BMC were similar. Sorted CFU-S-enriched
80-fold were found to have an f-factor of 0.1, while unfractionated control BMC were found to have an f-factor of 0.13
(Table 2). The difference in results is not statistically
significant.
Continued donor cell productivity. SBMC preparations
were also assayed for their ability to provide radioprotection
and survival in LI mice. BDFl mice received lethal irradia-
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TWO-DYE SORTING AND TRANSPLANTATON OF HSC
Figl.
1243
Sdoctionofbbstr~
gion of L S M - w m t o d BMC.
Tho a W s w roproaona right
anglo acattor; the ordinate. forward acattor. Tho box IRogion
1) thus s e l m s cells of low
atructural compkxiy and Iargor
sizo, io, tho bbst rogim. which
in thia oxporimont containod
10.9% of the cella roproaontod
in tho acattrgrem. Unita aro
arbitrary but linoor in all figures.
tion ( 1 1 .O Gy) before transplantation of 150 or 300 SBMC.
Seventy-seven percent of the mice receiving 150 SBMC and
100% of the mice receiving 300 SBMC survived beyond 60
days (Table 3). At 9 months posttransplantation, their white
blood cell counts, hematocrits. and differential blood smears
were normal, and femoral bone marrow from the lower dose
recipients contained reduced numbers of CFU-S(8) and
CFU-S( 12) (Table 4). similar to those found in primary
recipients of 1.5 x IO' to 3 x 106 normal donor BMC, as
previously r~ported.'~
Also. at 9 months. only male (donor)
Fig2.
Ano)y.band-
tkn of low Hoochst-contalnlng
d h in th. Mat r o g h IOh o d in Fig 1. Tho abaciroprosmta g m n (HMchst) fluIntonsky: tho ordinat0
roproucm coil numbor. Rogion
1 roproamta and a d m a 3.1%
of tho cells in tho histogram.
IRogion 2 roprerontr 3.0% of
tho coils, but m a found to contoin fm vioM0 colls and waa
diacardod).
or..~-
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McALISlER ET AL
1244
1
Fig3. A ~ l @ ~ e n d &
tion of high WGA-TR offinity
mils from Rogion 1 (low Hoochst-comeining cells) in Fig 2.
The eb8cis.s repreamit. red
(TR) fluorescent intensity: the
ordinate represents cell number. Region 1 represents end
selects 70.3% of the cells in the
histogram.
80
40
karyotypes were found in 25 consecutively examined chromosome spreads from the femoral marrow of each of five of the
above female recipients of 150 male SBMC.
Radioprotective ability and competitive status. Four of
the above mentioned primary recipients reconstituted with
300 male SBMC were killed at 1 year posttransplantation.
and LI female secondary recipients were given low doses ( 1
to 5 x IO’) of unsorted pooled femoral bone marrow cells
from these mice to test the radioprotective ability (RPA) of
progeny of the selected cells. The 30-day survival of these
secondary recipients was 100% ( IO of IO) for both cell doses
above I x IO5BMC and 78% for I x IO’ BMC (Table 3).
BMC from primary recipients reconstituted with 300 male
SBMC were combined with and competed against normal
(not previously transplanted) female BMC in a CFU-S( 12)
ratio of 4060 (as established by CFU-S assay of the BMC
preparations). Karyotyping of male and female secondary
TeMo 2. f-Fmor Dotomin”
in CFU-S(8)
recipient marrow was assessed at 45 and at 90 days. The
results in Table 5 indicate that only about 5% of the actively
dividing recipient BMC were from the sorted, previously
transplanted source. It is also clear from the data in Table 5
that the CFU-S(12) content of the BMC from 300 SBMC
recipients taken I5 months postransplantation was approximately 20% of the not previously transplanted normal RMC
(NBMC) value.
Morphology. The morphology of the stem cells stained
with Wright-Giemsa was observed under 1.OOOx magnification. The sorted cells were of a consistent morphology, which
fit previous descriptions.16 They appeared as small- to medium-sited undifferentiated lymphocytes about 7 to 8 pm in
diameter. They contained intensely basophilic nuclei: unTOM.3. 60-Day Sutvhml of Lotholly IrrediotedBDFl Mico
ReceMng Low Doses of SBMC
Tmolancl
Dore
Primay
300 SBMC
150 SBMC
Enrichmsclt.
103
103
No.Mio
DMhr
%suvM
13
13
0
3
100
77
9
11
10
2
0
0
78
100
100
-*t
i x 10)
3 x 10’
5 x 10’
~~
~~
*The dilution “ c t i o n facta for control recipients is based on the
ratio of cdl dow used in tho qoup versus that used in primay r @ i t s
of the test goup. The dilution carection facta for the test goup is b d
on the use of 1/3 of e primay recipiit spleen content per soconday
r@it.
tEnrichmentfor this expaiment was 80-foId far CFU-38).
$!%conday recipients received 1/3 minced d w n equiMbnt from
primw 3-how pwsege recipients.
*Enrichment based on day 8 spleen colony counts in fou mcipiits
from each cdl dose goup. The 1 5 M l dow mice whoso EM w a s
kaVotVpicelly examined for mab Manor) versus h o b h c i p i i t )
chromosome markersdisplayed only donor-type cdls 9 monthsposmasplontation. Three hundred SBMC primay recipiient monow was also
tested f a kayotype of CFU-S(l2) at 15 months p o s t t r a ~ l a n t(we
footnote ”*“ in Tebb 5).
tone f ” a l
content from eech of 4 d”was combined. Seconday transplont of 300 SBMC primay mipiits a 13 months eftor
primay transplant.
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TWO-DYE SORTING AND TRANSPLANTATION OF HSC
1245
Table 4. Blood and Bone Marrow Values of Recipients of Low
Doses of SBMC Measured 9 to 12 Months Posttransplantation
Type and
Group
N
WBC
(xlO-')
Hematccrit
Blood
6 44.7 f 0.1
150 SBMC
300 SBMC
2 46.0 i 2.0
Normal controls 10 48.6 f 0.6
5.6 f 1.1 L-80. G-18, M-2
11.6 f 4.6 L-85,612, M-3
5.5 i 0.3 L-73, G24, M-3
CFU-S/5 x
BMC ( X
Femur
Bone marrow
150 SBMC
Differential
lo4BMC
Day 12
Day 8
5 1.64 f 0.1
8.2 f 1.1
(n = 10)
ND"
300 SBMC
6 1.79 f 0.13
11.O f 0.7
Normal controls 10 2.10 f 0.2
5.8 f 0.7
(n = 12)
ND*
11.5 k 0.6
Values are mean f SE.
Abbreviations: L, lymphocytes; G, granulocytes; M, monocytes; ND,
no data.
"Marrow was used for secondary recipient survival study.
evenly stained, often indented, and surrounded by a thin
layer of cytoplasm.
DISCUSSION
Purification of hematopoietic stem cells is an important
step in the direction of better characterization of the stem
cell and its use in related research. The successful combination of the simple techniques".I2 that we used in combination
Table 5. Competition of Once-Transplanted SBMC With NBMC
Donor Groups
BMC transplantation
dosage
CFU-S(l2) content of
BMCt
CFU-S( 12) transplantation
ratio
45-day recipient karyotype ratio$
90-day recipient karyctype ratio$
1.5 x
lo6
92 f 3.9
0.5 x
lo6
148 f 8.0
38%
62%
6% f 2.6%
94% f 2.6%
* 1.3%
96% f 1.3%
4%
BMC of male karyotype taken from four female 300 SBMC primary
recipients (15-month survivors) were competed against fresh NBMC
(female) by combining the two donor cell suspensions and injecting them
into both male and female LI (1 1.O Gy) recipients. Karyotypes of the latter
recipients' 8MC were assessed at 45 days and at 9 0 days. Where
applicable, values are mean f SE.
*The presence of the SBMC (male) karyotype of the female primary
recipient donors for the competition experiment was confirmed by
reading 3 0 consecutive chromosome spreads in each of 20 day-12
spleen colonies from four female secondary recipients injected with only
this cell suspension.
tLow-dose separate aliquots of the competing BMC suspensions were
assayed in opposite sex recipients, and CFU-S(l2) contents of the
competing doses were calculatedfrom these results.
$Karyotype ratios for dividing BMC into recipients were determined at
45 days in two males and two females, and at 9 0 days in two males and
three females from among the recipients of the combined 8MC. The male
karyotype was not seen with any greater frequency in male recipients
than in female recipients.
provided reliably high levels of enrichment needed for
further characterization of the stem cell with a single passage
of cells through the FACS.
The methods used to isolate the stem cells directly reveal
some of the characteristics of the stem cell. Density sedimentation relies largely upon the density of those cells that arrest
in a given position in the medium. This low density in a
medium-sized cell cohort, which includes largely CFU-S and
cells that provide radiation survival, might reflect the status
of the cytoplasmic constituents. A lack of cytoplasmic
constituents, such as mitochondria and structural and functional cytoplasmic proteins, is consistent with the role of the
stem cell as a quiescent
The low Hoechst dye uptake
is likely to be the result of low cell membrane permeability to
the dye." Such low permeability might be expected of a cell
in Gostatus whose metabolic needs and receptivity to stimuli
should be minimal. Another perhaps less likely hypothesis is
that the stem cell DNA is inaccessible for Hoechst dye
binding. The WGA affinity of the cell is a function of the
presence of N-acetyl-d-glucosamine in glycoproteins of the
cell membrane" and is not restricted to stem cells.
The characteristics of purified hematopoietic stem cells
should match those already attributed to stem cells. Our
findings were that the morphology of sorted preparations was
uniform and matched that of already published descriptions.16
The f-factor for day 8 colonies was similar for purified and
unpurified bone marrow cells, indicating that the sorting
procedure did not affect such seeding. The ability to reconstitute the hematopoietic system of an irradiated mouse,
allowing its survival, is perhaps the most important functional definition of the pluripotent hematopoietic stem cell.
The ability of 150 purified stem cells to produce 77% survival
in LI mice, to return their constituent blood cell levels to
normal, and to remain the sole source of hematopoiesis 9
months later indicates the presence of primitive pluripotent
hematopoieticstem cells, sometimes referred to as "pre-CFUS,"20
at high levels of purification. Still more important is the
ability of such transplanted marrow to repopulate the hematopoietic system and produce survival in secondary
recipient^.^*^^^^^ We have shown this ability in the 30-day
survival of secondary recipient mice receiving as few as 1 x
lo5 unsorted BMC from primary recipients of 300 SBMC.
However, the once transplanted marrow from these same
primary recipients had lower than normal CFU-S content
and did not compete well against fresh unsorted BMC,
showing the same decline in potency with transplantation as
does unsorted BMC."
Recent reports indicate that the stem cell necessary for
long-term bone marrow repopulation is not necessarily selected with the CFU-S(8) or CFU-S(12) compartment in
multiparameter cell ~ o r t i n g . ~ In
, ~ 'our
, ~ ~preparations, stem
cells capable of producing long-term survival upon transplantation were selected in the same fraction, at least 20% to 40%
pure for CFU-S(8) and CFU-S(14). It is possible that the
two methods of cell sorting selected the subpopulations
(CFU-S and pre-CFU-S) by different criteria, which resulted in separation of subpopulations with these respective
attributes in one case and nonseparation in the other. It is
also possible that there is an overlap of characteristics in a
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
McALlSTER ET AL
1246
continuum of differentiating stem cells. These may range
from those originating short-lived day 8 spleen colonies23to
those individually responsible for long-term clones of both
myeloid and lymphoid cells.24In any event, it would appear
that long-term survival by primary recipients and their
maintenance of the ability to produce cells capable of further
transmitting survival is the capacity to be sought after in a
sorted hematopoietic stem cell preparation.
Enrichment results for day 14 colonies are subject to
cautious interpretation, since there is evidence that many
such late-appearing colonies may come from daughter
CFU-S and, as such, cannot be correlated one-to-one with
injected stem cell^.^.'^ When the CFU-S( 14):CFU-S(8) ratio
for sorted populations is compared with that for NBMC, the
ratios are generally 2 to 3 times greater in the sorted
populations (Table 1). However, if the large colonies (greater
than 2 mm diameter) present at day 14 are seen as representing continuing day-12 CFU-S and only those are counted,
the enrichment of large-colony CFU-S( 14) is more similar to
that for CFU-S(8), and the ratio is more similar to that for
unsorted donor BMC (Table 1). This indicates that the stem
cells from which the late-appearing small colony formers are
derived are either more numerous in the SBMC or more
active in producing daughter colonies. If the latter case is
true, the number of small day-14 colonies may provide an
approach to estimating the number of “pre-CFU-S” in
addition to recipient survival.
Weissman et a1 have recently published a method for
obtaining highly purified populations of murine CFU-S and
life-preserving cells using subsets of antibodies to cell surface
antigens in multiparameter
Their method should
be compared and combined with the studies using dyes for
provision of a rapid, inexpensive and highly enriched means
to purify hematopoietic stem cell populations. The possibility
of extending the dye absorption results to work with human
BMC should be explored for transplantation purposes and as
a means for providing information about the physiologic
nature of these cells.
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1990 75: 1240-1246
Transplantation of hematopoietic stem cells obtained by a combined
dye method fractionation of murine bone marrow
I McAlister, NS Wolf, ME Pietrzyk, PS Rabinovitch, G Priestley and B Jaeger
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