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HEMATOPOIESIS
Marked mitochondrial DNA sequence heterogeneity in single CD34⫹
cell clones from normal adult bone marrow
Myung Geun Shin, Sachiko Kajigaya, J. Philip McCoy Jr, Barbara C. Levin, and Neal S. Young
Somatic mitochondrial DNA (mtDNA) mutations accumulate with age in postmitotic tissues but have been postulated to
be diluted and lost in continually proliferating tissues such as bone marrow (BM).
Having observed marked sequence variation among healthy adult individuals’ total BM cell mtDNA, we undertook analysis
of the mtDNA control region in a total of
611 individual CD34ⴙ clones from 6 adult
BM donors and comparison of these results with the sequences from 580 CD34ⴙ
clones from 5 umbilical cord blood (CB)
samples. On average, 25% (range, 11% to
50%) of individual CD34ⴙ clones from
adult BM showed mtDNA heterogeneity,
or sequence differences from the aggregate mtDNA sequence of total BM cells of
the same individual. In contrast, only 1.6%
of single CD34ⴙ clones from CB showed
mtDNA sequence variation from the aggregate pattern. Thus, age-dependent accumulation of mtDNA mutations appears
relatively common in a mitotically active
human tissue and may provide a method
to approximate the mutation rate in mam-
malian cells, to assess the contribution of
reactive oxygen species to genomic instability, and for natural “marking” of hematopoietic stem cells; our data also have
important implications for the aging process, forensic identifications, and anthropologic conclusions dependent on the
mtDNA sequence. (Blood. 2004;103:
553-561)
© 2004 by The American Society of Hematology
Introduction
Mitochondrial DNA (mtDNA) comprises 0.1% to 1.0% of the total
DNA in most mammalian cells, and 2 to 10 copies come packaged
in each nucleated cell in each of up to 1000 mitochondria. Human
mtDNA is a double-stranded, maternally inherited circular molecule of 16 569 base pairs; its 37 genes encode for polypeptides of
the mitochondrial electron transport chain, transfer RNAs, and
ribosomal RNAs necessary for their synthesis.1,2 In comparison
with the nuclear genome, mtDNA has a modified genetic code,3 a
paucity of introns, and lack of histone protection.4 Past evidence
had indicated that mtDNA repair capacity was limited and that the
proximity of mtDNA to sites of reactive oxygen species generation
suggested that mtDNA may be more susceptible to mutation than
nuclear DNA. Although the limited repair capacity hypothesis has
been validated experimentally in some experimental systems,
recent data have shown that base excision repair mechanisms do
occur in mammalian mtDNA.5,6 Another major difference between
mtDNA and nuclear DNA is that multiple species of mtDNA can
coexist in a single cell, a condition called heteroplasmy. During
development and with aging, at least in nonmitotic tissues of the
neuromuscular system, mtDNA mutations not only accumulate but
achieve homoplasmy within tissues, perhaps as a result of random
genetic drift or of a selective replicative advantage conferred on 1
of the 2 heteroplasmic species.7
Because of its abundance and inherent variability, mtDNA has
been widely used for forensic identification and in anthropologic
studies. Furthermore, several hundred human diseases have been
associated with maternally inherited specific deletions and muta-
tions.8 Somatically acquired mtDNA mutations also have been
linked to aging and degenerative diseases, cancer, and autoimmunity.9 A large deletion of mtDNA is a hallmark of Pearson
syndrome, a constitutional disorder that includes sideroblastic
anemia.10 mtDNA mutations recently were reported also in apparently acquired sideroblastic anemia and in myelodysplastic syndromes in general.11 While we were unable to confirm these results
by amplification and direct sequencing of the entire mtDNA
genome in patients and healthy controls, we coincidentally observed numerous sequence changes in bulk samples of bone
marrow cells from our healthy controls as well as in patients in
these experiments.12 While mtDNA changes have been postulated
to underlie the aging process,13 mutant mtDNA genomes also have
been assumed to be lost by dilution from rapidly dividing tissues
such as bone marrow.8 We therefore undertook investigation of the
possibility that mtDNA mutations might accumulate with aging in
individual human CD34⫹ cells by examining a portion of mtDNA
thought to have a high rate of somatic mutation.14
From the Hematology Branch, Flow Cytometry Core Facility, National Heart,
Lung, and Blood Institute, National Institutes of Health, Bethesda, MD; and
Biotechnology Division, Chemical Science and Technology Laboratory,
National Institute of Standards and Technology, Gaithersburg, MD.
M.G.S. and S.K. contributed equally to this work.
Submitted May 29, 2003; accepted September 11, 2003. Prepublished online
as Blood First Edition Paper, September 22, 2003; DOI 10.1182/blood-200305-1724.
BLOOD, 15 JANUARY 2004 䡠 VOLUME 103, NUMBER 2
Materials and methods
Normal bone marrow and cord blood
Bone marrow (BM) specimens from 6 healthy adult donors were collected after
informed consent was obtained following protocols approved by the Institutional
Review Board of the National Heart, Lung, and Blood Institute. Of 5 cord blood
(CB) samples, 2 came from the Blood Bank at the National Institutes of Health
(Bethesda, MD) and 3 from the New York Blood Center (New York, NY).
Reprints: Neal S. Young, Bldg 10, Rm 7C103; NIH; 9000 Rockville Pike,
Bethesda, MD 20892-1652; e-mail: [email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2004 by The American Society of Hematology
553
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554
SHIN et al
Single CD34ⴙ cell sorting
Mononuclear cells from BM and CB were separated by density gradient
centrifugation and washed twice in phosphate-buffered saline (PS). The
number of cells suspended in PBS was adjusted to 2 ⫻ 107 cells per
milliliter. Ten microliters of anti-CD34 phycoerythrin (PE)–conjugated
antibody (BD Bioscience, Franklin Lakes, NJ) were added to each 12 ⫻ 75
mm tube containing 100 ␮L cell suspension. After incubation for 30
minutes at 4°C, cells were washed using cold PBS and resuspended in 0.5
mL buffer. Cell sorting was performed on a MoFlo Cytometer (DakoCytomation, Ft Collins, CO) using 100 mW of the 488 nm line of an argon
laser (I-90, Coherent, Palo Alto, CA) for excitation. Forward scatter was the
triggering parameter. Fluorescence of PE was detected using a 580/30
bandpass filter. Single cell deposition was accomplished using the CyClone
automated cloner (Dako-Cytomation); in the 0.5 single drop mode and with
gating based on forward scatter and PE fluorescence, individual CD34⫹
cells were placed into each well of a 96-well plate containing 100 ␮L
culture media (Figure 1A).
BLOOD, 15 JANUARY 2004 䡠 VOLUME 103, NUMBER 2
without DNA templates, were subjected to the same PCR amplification conditions and in all cases confirmed to be negative. To prevent DNA crosscontamination, special precautions were taken for each procedure of cell harvest,
DNA extraction, PCR amplification, and DNA sequencing.
Sequence analysis of the mtDNA control region
We amplified and then directly sequenced the 1121–base pair control region
(nucleotides 16 024 to 16 569 and 1 to 576) (Figure 1B) using the BigDye
Terminator v3.0 Ready Reaction kit (Applied Biosystems, Foster City, CA)
and the ABI Prism 3100 Genetic Analyzer (Applied Biosystems). The
following oligonucleotide primers were used for sequencing: 5⬘-CAGTGTATTGCTTTGAGGAGG-3⬘, 5⬘-CATCTGGTTCCTACTTCAGGGTC-3⬘,
5⬘-TTAACTCCACCATTAGCACC-3⬘, 5⬘-GCATGGAGAGCTCCCGTGAGTGG3-3⬘, 5⬘-CACCCTATTAACCACTCACG-3⬘, and 5⬘-TACATTACTGCCAGCCACCATG-3⬘. mtDNA sequences experimentally obtained were compared with the Revised Cambridge Reference Sequence
Culture and harvest of CD34ⴙ cells
Individual CD34⫹ cells placed into separate wells of 96-well plates were cultured
in serum-free medium containing 100 ng/mL stem cell factor (SCF), 100 ng/mL
Flt-3, 100 ng/mL thrombopoietin (TPO), with or without 50 ng/mL granulocyte
colony-stimulating factor (G-CSF) (all from Stem Cell Technologies, Vancouver,
BC, Canada). After culture for 5 days, each well of the microtiter plate was
carefully observed using an inverted microscope (Olympus IX50, Olympus,
Melville, NY) to determine growth and plating efficiency of single CD34⫹ cells
and to grade growth with the following scoring system based on cell number in
each CD34⫹ clone: grade 1, 5 or fewer cells per well; grade 2, 6 to 10 cells per
well; grade 3, 11 to 20 cells per well; grade 4, 21 or more cells per well (Figure
1C). Plating efficiency was defined as the number of positive (cells were present)
wells divided by total wells ⫻ 100. Each CD34⫹ clone was harvested from the
well by vigorous pipetting and dispensed into a 1.5 mL microcentrifuge tube and
rinsed with 200 ␮L PBS. Cells were collected after centrifugation at 300g for 5
minutes and then washed with PBS. Cell pellets were stored at ⫺80°C.
DNA extraction from individual CD34ⴙ clones
A total of 30 ␮L of 1 ⫻ Tris-EDTA (TE) buffer was placed in each 1.5 mL
tube containing a cell pellet. The cells were lysed by incubation at 95°C for
10 minutes with occasional shaking to liberate the total DNA. The lysate
was briefly microcentrifuged and stored at ⫺20°C.
PCR amplification of the mtDNA control region
Cell lysates of individual CD34⫹ clones were subjected to amplification of
mtDNA using the long-and-accurate polymerase chain reaction (LA PCR) kit
(TaKaRa LA Taq, Panvera, Madison, WI). Two-step PCR amplification was
performed with outer and inner pairs of primers to generate sufficient template
from CD34⫹ clones for sequencing of the mtDNA control region. The outer pair
of primers (5⬘-CGCCTACACAATTCTCCGATC-3⬘ and 5⬘-ACTTGGGTTAATCG TGTGACC-3⬘) was used for amplification of the fragment spanning
nucleotides 15 574 to 16 569 and 1 to 921 of the revised human mtDNA Revised
Cambridge Reference Sequence. The inner nested pair of primers (5⬘TTAACTCCACCATTAGCACC-3⬘ and 5⬘-GAAAGGCTA GGACCAAACCTA-3⬘) amplified the fragment spanning nucleotides 15 971 to 16 569 and 1 to
670 (Figure 1B). The primary PCR mixture contained 400 ␮M of each
deoxyribonucleoside triphosphate (dNTP), 2 units of LA Taq (TaKaRa LA Taq),
0.8 ␮M outer primers, and 3 ␮L cell lysate in a total volume of 30 ␮L. PCR
amplification was carried out in a thin-wall 0.5 mL PCR tube using the DNA
thermal cycler 9700 (Perkin-Elmer, Foster City, CA): 1 cycle of 96°C for 1
minute; then 35 cycles of 94°C for 30 seconds, 52°C for 50 seconds, and 72°C for
1 minute with a 10-second increase per cycle; ending with 1 cycle of 72°C for 5
minutes. The secondary PCR was performed in 50 ␮L reaction mixture
containing 400 ␮M of each dNTP, 2 units of LA Taq, 0.8 ␮M inner nested
primers, and 2 ␮L primary PCR product under the same amplification conditions
as described above. Secondary PCR samples were electrophoresed on 1%
agarose gels and stained with ethidium bromide to assess the purity and size of
the DNA fragments and subsequently purified using the QIA quick PCR
purification kit (Qiagen, Valencia, CA). The negative controls, reaction mixtures
Figure 1. Flow chart, linearized map of mtDNA control region, and single CD34ⴙ
clones. (A) Mononuclear cells from BM and CB were separated by density gradient
centrifugation and washed twice in phosphate-buffered saline. CD34⫹ cells were sorted by
single cell deposition into 96-well microtiter plates using a phycoerythrin anti-CD34
monoclonal antibody (BD Biosciences), a MoFlo cytometer, and a CyClone automated
cloner (Dako-Cytomation) in the 0.5 single drop mode. After 5 days of culture in media
containing stem cell factor, Flt-3, thrombopoietin, and G-CSF, each well of the microplate
was carefully examined and scored for cell number. To directly sequence the control region
of mtDNA, DNA in these colonies, derived from single CD34⫹ cells, was subjected to
nested gene amplification (see “Materials and methods”). Sequencing was performed on
an ABI Prism 3100 Genetic Analyzer in both orientations. Evidence of mtDNA heterogeneity was further confirmed by reamplification of the original lysate. (B) Linearized map and
function location of mtDNA control region between nucleotides 16 024 to 16 569 and 1 to
576 (D-loop); HV1 (hypervariable segment 1, nucleotides 16 024 to 16 383), HV2
(hypervariable segment 2, nucleotides 57 to 372), OH (H-stand origin, nucleotides 110 to
441), CSB (conserved sequence block, nucleotides 213 to 235, nucleotides 299 to 315,
nucleotides 346 to 363), mt5 (control element, nucleotides 16 194 to 16 208), mt3L
(L-strand control element, nucleotides 16 499 to 16 506), TAS (termination-associated
sequence, nucleotides 16 157 to 16 172), PL (L-strand promoter, nucleotides 392 to 345),
PH1 (major H-strand promoter, nucleotides 545 to 567), TFB (mitochondrial transcription
factor binding site, nucleotides 233 to 260, 276 to 303, 418 to 445, 523 to 550), mt4H
(H-strand control element, nucleotides 371 to 379), mt3H (H-strand control element,
nucleotides 384 to 391), and 7S DNA (nucleotides 16 106 to 16 191). The homopolymeric
C tracts located on HV1 (nucleotides 16 184 to 16 193; 5CT4C) and HV2 (nucleotides 303
to 315; 7CT5C). (C) The number and morphology of CD34⫹ clones after 5-day suspension
culture. Each picture (original magnification, ⫻ 200) shows a different grade: (i) 5 or fewer
cells per well, grade 1; (ii) 6 to 10 cells per well, grade 2; (iii) 11 to 20 cells per well, grade 3;
and (iv) 21 or more cells per well, grade 4.
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BLOOD, 15 JANUARY 2004 䡠 VOLUME 103, NUMBER 2
mtDNA HETEROGENEITY IN SINGLE CD34⫹ CLONES
555
Table 1. Plating efficiency and grade of CD34ⴙ clones from normal adult bone marrow and cord blood samples after 5-day culture
Grade
1
2
Age, y/sex
ⴙ
ⴚ
1
47/F
14
19
2
38/F
27
22
3
43/M
103
4
34/M
5
54/M
6
34/F
Donor
ⴙ
3
4
Subtotal no.
ⴚ
ⴙ
ⴚ
ⴙ
ⴚ
ⴙ
ⴚ
9
12
16
14
36
2
75
1
2
1
3
6
0
35
176
65
84
49
42
122
5
45
71
33
74
50
53
112
98
208
67
110
60
59
135
82
131
43
103
79
70
150
627
218
385
255
241
561
Microplate*
Plating Efficiency, %
Total no.
ⴙ
ⴚ
ⴙ
ⴚ
47
122
2
2
39.1
24.5
27
62
5
5
7.3
5.6
339
307
646
10
10
35.3
32.0
21
240
219
459
10
10
25.0
22.8
5
360
382
742
10
10
37.5
39.8
16
354
320
674
10
10
36.9
33.3
49
1403
1302
2705
47
47
31.1
BM
BM subtotal
369
BM total
996
603
496
610
2705
94
28.9
30.0
CB
1
—
46
85
79
155
167
309
398
124
690
673
1363
10
10
71.9
70.1
2
—
8
8
6
21
23
78
369
309
406
416
822
5
5
84.6
86.7
3
—
28
55
33
45
57
44
280
245
398
389
787
5
5
82.9
81.0
4
—
23
30
21
26
30
59
346
299
420
414
834
5
5
87.5
86.3
5
—
22
33
23
36
29
52
286
243
360
364
724
5
5
75.0
75.8
127
211
162
283
306
542
1679
1220
2274
2256
4530
30
30
79.0
78.3
CB subtotal
CB total
338
445
848
2899
4530
60
78.6
PhE indicates plating efficiency (see “Materials and methods”); grade 1, 5 or fewer cells per well; grade 2, 6 to 10 cells per well; grade 3, 11 to 20 cells per well; grade 4, 21
or more cells per well; ⫹, culture media containing 100 ng/m of each stem cell factor, Flt-3, thrombopoietin, serum-free media, and 50 ng/mL G-CSF; ⫺, same culture media
without G-CSF; BM, bone marrow; CB, cord blood; and —, not applicable.
*No. of 96-well microplates.
(http://www.mitomap.org)2 using the BLAST2 program (http://www.ncbi.
nlm.nih.gov/blast/bl2seq/bl2.html) and the database search tool MitoAnalyzer (http://www.cstl.nist.gov/biotech/strbase/mitoanalyzer.html)15 to determine polymorphisms and mutations. All automated results were manually confirmed. To exclude potential artifacts, PCR amplifications from
original cell lysates were additionally replicated 1 or 2 more times, and
when nucleotide changes were reproduced in all independent PCR amplifications, they were considered to be confirmed. Several CD34⫹ clones with
potential mtDNA-nucleotide changes were eliminated because no mtDNA
was amplified when the PCR was replicated. The PCR replication probably
failed due to the low concentration of mtDNA.
TA cloning
In preliminary experiments, 285-bp amplicons were generated by gene
amplification of wild-type mtDNA and mtDNA altered in a single base and
then mixed in varying proportions. On sequencing of the mixtures, the
lower limit of detection of a minor species of mtDNA was approximately
20%. Mixed nucleotide signals on sequencing chromatograms, when
observed in the current study, were assumed to represent at least 20%
heteroplasmy but could also result from gene amplification artifacts. To
confirm heteroplasmy and mixed nucleotide signals in the sequences of the
mtDNA control region, PCR products were directly inserted into the pCR
2.1-TOPO vector and transformed into competent Escherichia coli (TOP10
cells) using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA). Recombinant plasmids isolated from 8 to 12 white colonies were sequenced.
Statistical analysis
A ␹2 test was used to determine statistical differences in the frequency of
heteroplasmy in adult bone marrow and cord blood. The 1-way analysis of
variance (ANOVA) test was performed to examine whether the number of
cells in each clone or the culture media produced significant statistical
differences in mtDNA heterogeneity; P ⬍ .05 was considered significant.
Table 2. Assayed number of individual CD34ⴙ clones from normal adult bone marrow and cord blood samples after 5-day culture
Grade
1
2
ⴙ
ⴚ
1
14
14
2
13
10
3
15
4
5
6
Donor
ⴙ
3
4
Subtotal no.
ⴚ
Total
assayed no.
73
42
115
21
15
36
70
50
120
14
57
54
111
5
67
47
114
15
59
56
115
41
347
264
611
ⴚ
ⴙ
ⴚ
ⴙ
ⴚ
8
12
16
14
35
2
1
2
1
3
6
0
15
15
15
15
15
25
5
13
13
14
14
15
13
15
12
14
15
13
15
15
25
14
11
15
15
15
15
15
77
68
71
77
75
121
ⴙ
BM
BM subtotal
81
BM total
158
139
152
162
611
CB
1
15
15
14
15
15
15
15
15
59
60
119
2
8
8
6
15
15
16
26
26
55
65
120
3
14
13
14
15
15
15
5
13
48
56
104
4
15
14
15
15
15
15
15
15
60
59
119
5
14
15
15
14
15
15
15
15
59
59
118
66
65
64
74
75
76
76
84
281
299
580
CB subtotal
CB total
131
Abbreviations are explained in Table 1.
138
151
160
580
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556
BLOOD, 15 JANUARY 2004 䡠 VOLUME 103, NUMBER 2
SHIN et al
Table 3. Nucleotide sequence changes of mtDNA control region
from aggregate cells
Table 3. Nucleotide sequence changes of mtDNA control region
from aggregate cells (continued)
Donor no. and polymorphism
Donor no. and polymorphism
Affected mtDNA gene
BM donor 1
Affected mtDNA gene
BM donor 6
73A⬎G
HV2, 7S
73A⬎G
HV2, 7S
150C⬎T
HV2, 7S, OH
150C⬎T
HV2, 7S, OH
263A⬎G
HV2, OH
263A⬎G
HV2, OH
8CT6C*
HV2, OH, CSB2
8CT6C*
HV2, OH, CSB2
16192C⬎T
HV1, 7S
517A⬎G†
16270C⬎T
HV1, 7S
16270C⬎T
HV1, 7S
16292C⬎T
HV1, 7S
16362T⬎C
HV1, 7S
BM donor 2
—
73A⬎G
HV2, 7S
185G⬎A
HV2, 7S, OH
263A⬎G
HV2, OH
73A⬎G
HV2, 7S
7CT6C*
HV2, OH, CSB2
146T⬎C
HV2, 7S, OH
195T⬎A
HV2, OH
A478G†
—
CB donor 1
16093T⬎C
HV1
263A⬎G
HV2, OH
16158A⬎T
HV1, 7S, TAS
7CT6C*
HV2, OH, CSB2
16172T⬎C
HV1, 7S, TAS
489T⬎C
16183A⬎C
HV1, 7S
514-515delCA
—
16189T⬎C (12C)*
HV1, 7S
16166A⬎T
—
16219A⬎G
HV1, 7S
16169delC†
HV1, 7S, TAS
16278C⬎T
HV1, 7S
16172T⬎C
HV1, 7S, TAS
16223C⬎T
HV1, 7S
BM donor 3
—
73A⬎G
HV2, 7S
16354C⬎T
HV1, CSB3
146T⬎C
HV2, 7S, OH
16519T⬎C
7S
152T⬎C
HV2, 7S, OH
195T⬎C
HV2, OH
72T⬎C
HV2, 7S
263A⬎G
HV2, OH
253C⬎T
HV2, OH, TFB1
8CT6C*, 9CT6C*
HV2, OH, CSB2
263A⬎G
HV2, OH
9CT6C*
HV2, OH, CSB2
514-515delCA
—
CB donor 2
16223C⬎T
HV1, 7S
10CT6C*
HV2, OH, CSB2
16278C⬎T
HV1, 7S
8CT6C*
HV2, OH, CSB2
16294C⬎T
HV1, 7S
16256C⬎T
HV1, 7S
16390G⬎G
7S
16298T⬎C
HV1, 7S
16519T⬎C
7S
BM donor 4
93A⬎G
HV2, 7S
CB donor 3
95A⬎C
HV2, 7S
73A⬎G
HV2, 7S
185G⬎A
HV2, 7S, OH
146T⬎C
HV2, 7S, OH
189A⬎G
HV2, 7S, OH
189A⬎G
HV2, 7S, OH
236T⬎C
HV2, OH
194C⬎T
HV2, OH
8CT6C*
HV2, OH, CSB2
195T⬎C
HV2, OH
247G⬎A
HV2, OH, TFB1
204T⬎C
HV2, OH
263A⬎G
HV2, OH
207G⬎A
HV2, OH
263A⬎G
HV2, OH
514-515delCA
—
16093T⬎C
HV1
279T⬎C
HV2, OH
16129G⬎A
HV1, 7S
8CT6C*
HV2, OH, CSB2
16148C⬎T
HV1, 7S
9CT6C*
HV2, OH, CSB2
16168C⬎T
HV1, 7S, TAS
16223C⬎T
HV1, 7S
16172T⬎C
HV1, 7S, TAS
16292C⬎T
HV1, 7S
16187C⬎T*
HV1, 7S
16519T⬎C
7S
16188C⬎G*
HV1, 7S
CB donor 4
16189T⬎C*
HV1, 7S
73A⬎G
HV2, 7S
16223C⬎T
HV1, 7S
249delA
HV2, OH, TFB1
16230A⬎G
HV1, 7S
290-291delAA
HV2, OH, TFB2
16278C⬎T
HV1, 7S
7CT6C*
HV2, OH, CSB2
16293A⬎G
HV1, 7S
489T⬎C
—
16311T⬎C
HV1, 7S
493A⬎G
—
16320C⬎T
HV1, 7S
514-515delCA
BM donor 5
—
16223C⬎T
HV1, 7S
73A⬎G
HV2, 7S
16298T⬎C
HV1, 7S
263A⬎G
HV2, OH
16325T⬎C
HV1, 7S
7CT6C*
HV2, OH, CSB2
16327C⬎T
HV1, 7S
16519T⬎C
7S
514-515delCA
—
16126T⬎C
HV1, 7S
16294C⬎T
HV1, 7S
16296C⬎T
HV1, 7S
16519T⬎C
7S
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BLOOD, 15 JANUARY 2004 䡠 VOLUME 103, NUMBER 2
Table 3. Nucleotide sequence changes of mtDNA control region
from aggregate cells (continued)
Donor no. and polymorphism
Affected mtDNA gene
CB donor 5
73A⬎G
HV2, 7S
150C⬎T
HV2, 7S, OH
195T⬎C
HV2, OH
263A⬎G
HV2, OH
7CT6C*
HV2, OH, CSB2
16171A⬎G
HV1, 7S, TAS
16172T⬎C
HV1, 7S, TAS
16189T⬎C*
HV1, 7S
16193C⬎C/CC*
HV1, 7S
16223C⬎T
HV1, 7S
16320C⬎T
HV1, 7S
16519T⬎C
7S
HV1 indicates hypervariable segment 1; HV2, hypervariable segment 2; 7S, 7S
DNA; OH, H-strand origin; CSB2, conserved sequence block II; TAS, terminationassociated sequence; TFB1, mitochondrial transcription factor 1 binding site; and —,
not applicable.
*Homopolymeric C tracts at nucleotides position at 303 to 315 (for example,
8CT6C defined CCCCCCCCTCCCCCC) and 16184 to 16193 in HV2 and HV1,
respectively.
†New mtDNA polymorphisms (not listed in accepted database).
Results
Culture of single CD34ⴙ cells
After sorting, single CD34⫹ cells were cultured in individual wells of
96-well plates in serum-free medium containing selected hematopoietic
growth factors, with or without G-CSF. Plating efficiency was microscopically determined by the presence of clusters of viable cells. CD34⫹
cell-derived colonies were classified according to the cell number per
well (Figure 1C). Although there was some variation of plating
efficiency of CD34⫹ cells among 6 healthy BM and 5 CB donors,
overall average plating efficiency in BM and CB was 30%
(30.0% ⫾ 11.7%, mean ⫾ SD) and 79% (78.6% ⫾ 11.7%), respectively (Table 1) and was not affected by G-CSF. As expected, the colony
size was increased in the presence of G-CSF.
mtDNA control region
To assess heterogeneity of the mtDNA sequences among CD34⫹ cells
from healthy BM and CB donors, we targeted the 1121 bp of mtDNA
control region known to contain multiple mutational hotspots.14 More
than 100 CD34⫹ clones per each donor except for donor no. 2 were
subjected to sequencing analysis, resulting in a total number of 611 and
580 CD34⫹ clones from adult BM and CB, respectively (Table 2).
Aggregate genotype of the mtDNA control region from
total BM and CB cells
To identify mtDNA heterogeneity in individual CD34⫹ clones, we first
determined the aggregate genotype of total BM cells from each donor.
There was marked variation in the number of nucleotide changes among
individual healthy BM donors, with ranges of 6 (donor no. 1) to 23
(donor no. 4) (11.3 ⫾ 6.1, mean ⫾ SD) (Table 3). A total of 68 mtDNA
sequence variants were found in aggregate BM cells from 6 healthy
donors; among these 66 variants were already listed in the polymorphism database (http://www.mitomap.org). Two new nucleotide
variants were classified mutations (A478G and A517G in donor
nos. 2 and 6, respectively). The number of mtDNA sequence
changes from aggregate CB cells also showed individual
variations (Table 3) but, interestingly, 3 (CB nos. 2, 3, and 5) of
mtDNA HETEROGENEITY IN SINGLE CD34⫹ CLONES
557
5 CB donors had length variations of poly C tract at nucleotide
position 303 to 315 and 16 183 to 16 193.
mtDNA heterogeneity among individual CD34ⴙ clones
Analysis of 611 CD34⫹ clones from the 6 healthy BM donors revealed
that a total of 152 clones (24.9% ⫾ 17.2%, mean ⫾ SD) displayed
mtDNA heterogeneity distinct from the donor’s corresponding aggregate mtDNA sequences (Table 4). Common patterns of mtDNA
heterogeneity in CD34⫹ clones among 6 BM donors were 1 or 2
nucleotide changes (substitution, insertion, or deletion) in addition to the
polymorphisms detected in the respective aggregate mtDNA. Among
them, most differences were due to single nucleotide substitutions at
various positions and length alterations in the poly C tract localized
between nucleotides 303 to 315 (Figure 2B). The heterogeneous
mtDNA of CD34⫹ clones in 6 BM donors was classified into several
unique patterns according to nucleotide changes; 8, 5, 7, 6, 14, and 6
patterns in donor nos. 1 to 6, respectively (Table 4). The mean
proportion of unique heterogeneous pattern of mtDNA among
single CD34⫹ clones was 7.5% (7.5% ⫾ 3.3%) (Table 5). Figure
2A disclosed one of the typical heteroplasmic mutations in
CD34⫹ clone derived from BM donor no. 6: 200A⬎G/A
heteroplasmy in one CD34⫹ clone of adult BM donor no. 6 was
clearly identified using TA cloning. Neither the presence of
G-CSF nor the colony size was statistically correlated with the
proportion of CD34⫹ clones with variant mtDNA (Table 6).
Only 9 clones of 580 CD34⫹ clones (1.6%, 1.6% ⫾ 1.5%) from the
5 CB donors showed mtDNA heterogeneity distinct from the sequences
of the donor’s corresponding aggregate mtDNA as well as from other
CD34⫹ clones (Table 5). The mean proportion of unique mtDNA
heterogeneity pattern among single CD34⫹ clones from CB was 1.2%
(1.2% ⫾ 1.0%). As noted in a single CD34⫹ clone from BM donor no.
2, the mtDNA sequence of a single CD34⫹ clone from CB donor no. 1
showed an extremely distinct pattern as compared with the aggregate
CB mtDNA and other CD34⫹ clones (Table 4).
Characteristics of CD34ⴙ clones derived from adult BM and CB
Genetic changes in normal marrow and cord blood samples are
summarized in Table 5. Most striking, about a quarter of the more than
600 CD34⫹ cell clones of adult bone marrow differed from the
aggregate mtDNA sequence of each specific donor while, in contrast,
under 2% of the almost 600 cord blood CD34⫹ cell clones were
different from their respective individual aggregate sequence. The
differences for unique clones (different from aggregate sequence and
other CD34⫹ cell clones) were also marked. No solitary nucleotide
substitutions were observed in CB mtDNA CD34⫹ clones.
Hotspots of the mtDNA control region in CD34ⴙ clones
from adult BM
As anticipated, a high incidence of nucleotide variations was observed in
both HV2 (110 of 152 total genetic changes observed, or 72%) and HV1
(12 of 152 total genetic changes, or 7.9%) segments; most mutations
were localized in the HV2 homopolymeric C tracts between nucleotides
303 and 315 (43%, or 66 of 152) (Figure 2B).
Discussion
As we previously observed, a wide range of polymorphisms and
mutations not previously described in accepted databases were noted
among healthy individuals in the aggregate genotype (the sequence
obtained from total BM cells), even in this relatively short region of
mtDNA. When more than 600 CD34⫹ cell clones from 6 adult BMs were
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558
BLOOD, 15 JANUARY 2004 䡠 VOLUME 103, NUMBER 2
SHIN et al
Table 4. Summary of mtDNA heterogeneity among individual single CD34ⴙ clones from normal adult bone marrow and cord blood
Heterogeneity
Different
Donor and mtDNA sequence
BM donor 1
“Aggregate” sequence of total BM cells
Nonaggregate sequences
⫹8CT6C*, 9CT6C*
⫹9CT6C*
⫹7CT6C*
⫹189A⬎G/A
⫹204T⬎C
⫹277C⬎T
⫹514insCA
⫹16114C⬎T
BM donor 2
“Aggregate” sequence of total BM cells
Nonaggregate sequences
⫹16184C⬎CC (11C)*
⫹16131T⬎C/T
⫹16145G⬎A
⫹16184C⬎CCCC (13C)*
73A⬎G, 263A⬎G, 191A⬎AA, 194C⬎T, 199T⬎C, 207G⬎A, 8CT6C*,
489T⬎C, 16147C⬎T, 16173C⬎T, 16245C⬎T, 16362T⬎C
BM donor 3
“Aggregate” sequence of total BM cells
Nonaggregate sequences
⫹9CT6C*, 10CT6C*
⫹9CT6C*
⫹8CT6C*
⫹182C⬎T/C, 8CT6C*, 9CT6C*
⫹71delG, 9CT6C*, 10CT6C*
⫹279T⬎C/T
⫹16153G⬎A
BM donor 4
“Aggregate” sequence of total BM cells
Nonaggregate sequences
⫹8CT6C*, 9CT6C*
⫹514-515delCA, 514insCA†
⫹7CT6C*, 8CT6C*
⫹9CT6C*, 10CT6C*
⫹89T⬎C
⫹8CT6C*, 9CT6C*, 16093T‡
BM donor 5
“Aggregate” sequence of total BM cells
Nonaggregate sequences
⫹514insCA‡
⫹264C⬎T
⫹514-515delCA, 514insCA†
⫹264C⬎T/C
⫹7CT6C*, 8CT6C*
⫹146T⬎C, 514insCA‡
⫹146T⬎C, 264C⬎T/C
⫹146T⬎C/T, 514insCA‡
⫹146T⬎C
⫹189A⬎G
⫹264C⬎T/C, 514insCA‡
⫹161T⬎C/T, 264C⬎T, 514insCA‡
⫹16189T⬎C*
⫹16296C⬎C/T
BM donor 6
“Aggregate” sequence of total BM cells
Nonaggregate sequences
⫹8CT6C*, 9CT6C*
⫹200A⬎G/A
⫹200A⬎G
⫹200A⬎G/A, 7CT6C*, 8CT6C*
⫹200A⬎G/A, 8CT6C*, 9CT6C*
⫹7CT6C*, 8CT6C*
Unique
Clone no.
No.
%
No.
%
85
0
30
0
26.1
0
8
0
7.0
0
9
0
25.0
0
5
0
13.9
0
24
0
20.0
0
7
0
5.8
0
19
0
17.1
0
6
0
5.4
0
57
0
50.0
0
14
0
12.3
0
13
0
11.3
0
6
0
5.2
22
2
1
1
1
1
1
1
27
5
1
1
1
1
96
11
6
2
2
1
1
1
92
11
3
2
1
1
1
57
19
18
5
3
2
2
1
1
1
1
1
1
1
1
102
5
3
2
1
1
1
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BLOOD, 15 JANUARY 2004 䡠 VOLUME 103, NUMBER 2
mtDNA HETEROGENEITY IN SINGLE CD34⫹ CLONES
559
Table 4. Summary of mtDNA heterogeneity among individual single CD34ⴙ clones from normal adult bone marrow and cord blood
(continued)
Heterogeneity
Different
Donor and mtDNA sequence
CB donor 1
“Aggregate” sequence of total CB cells
73A⬎G, 153A⬎G, 183A⬎G, 263A⬎G, 7CT6C*, 8CT6C*, 325C⬎T,
Unique
Clone no.
No.
%
No.
%
118
1
0
1
0
0.8
0
1
0
0.8
120
0
0
0
0
100
0
4
0
3.8
0
2
0
1.9
118
1
0
1
0
0.8
0
1
0
0.8
115
0
3
0
2.5
0
3
0
2.5
463C⬎T, 485T⬎C, 489T⬎C, 514-515delCA, 16198T⬎C,
16223C⬎T, 16268C⬎T, 16354C⬎T, 16381T⬎A, 16519T⬎C
CB donor 2
“Aggregate” sequence of total CB cells
CB donor 3
“Aggregate” sequence of total CB cells
Nonaggregate sequences
⫹316T⬎G/C
⫹305C⬎A, 306C⬎A, 307C⬎A
CB donor 4
“Aggregate” sequence of total CB cells
⫹6CT6C*, 16022T⬎C
CB donor 5
“Aggregate” sequence of total CB cells
Nonaggregate sequences
⫹16184C⬎CCC (12C)*
⫹16189T⬎C*
⫹16184C⬎CCCC (13C)*
3
1
1
1
1
Different indicates different from aggregate cell sequence; unique, uniquely different heterogeneity; ⫹, mtDNA nucleotide changes in comparison with aggregate cell
mtDNA sequence.
*Homopolymeric C tracts at nucleotides position at 303 to 315 (for example, 8CT6C defined CCCCCCCCTCCCCCC) and 16184 to 16193 in HV2 and HV1, respectively.
†Mixed pattern of 514-515delCA and 514insCA.
‡The same as the Cambridge Reference Sequence but different from the aggregate sequence.
analyzed, we found marked sequence heterogeneity in all donors. In
comparison with each person’s aggregate BM mtDNA sequence, individual CD34⫹ cells showed sequence differences in 11% to 50% of the
CD34⫹ cell clones; sequence that was uniquely different from both the
aggregate sequence and the other cell clones was present in 5% to 14% of
the CD34⫹ cells. Approximately half the observed changes in individual
clones’ mtDNA sequence occurred in a homopolymeric C tract (nucleotides 303 to 315), and the remaining were point substitutions outside the
poly C region. Low levels of heteroplasmy (where the minor species is
less than 20% that of the major heteroplasmic species) may be technically
difficult to detect; nevertheless, we saw evidence of heteroplasmy in
sequence variants among individual adult CD34⫹ cells. Neither the
combination of growth factors utilized during the brief period of in vitro
culture nor the colony size correlated with the proportion of CD34⫹ clones
with variant mtDNA, suggesting that our observed results were not
secondary to the proliferation of cells during the few days of tissue culture.
Figure 2. mtDNA heterogeneity in individual CD34ⴙ
clones. (A) Sequence chromatogram with mixed nucleotide signal (G/A) at nucleotide position 200 in HV2 from 1
CD34⫹ clone of BM donor no. 6 (left), and then was
clearly divided into wild type (center) and mutation
(200A⬎G/A) (right) after TA cloning. (B) Poly C length
heteroplasmy between nucleotide position 303 and 315
from one of the CD34⫹ clones of BM donor no. 3 (left).
Reverse-sequence analysis after TA cloning revealed
poly C length heteroplasmy of 8CT6C (center) and
9CT6C (right).
The marked degree of sequence heterogeneity among individual
CD34⫹ cell clones was unexpected, because sequence variations in
mtDNA have not been thought to be sustainable in dividing tissues.
Because somatic mutations in general have been correlated with
aging of the organism, we next undertook similar experiments
utilizing CD34⫹ cells derived from CB. CB hematopoietic progenitor cells have a higher proliferative capacity compared with adult
CD34⫹ cells, as reflected in both better plating efficiency and large
colony size in tissue culture. Sequencing 580 clones from 5
samples of CB from different donors (Table 2) indicated that only
an average of 1.6% of the CD34⫹ clones had an mtDNA sequence
divergent from that of the aggregate CB (Tables 4 and 5).
The mtDNA control region contains many regulatory elements, some
of which are important in the initiation of transcription and replication of
mtDNA; control region mutants might alter the ratio of proteins or
transcripts derived from mtDNA to relevant nuclear DNA gene products,
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560
BLOOD, 15 JANUARY 2004 䡠 VOLUME 103, NUMBER 2
SHIN et al
Table 5. mtDNA heterogeneity characteristics of CD34ⴙ clones from adult BM and CB
Adult BM CD34ⴙ
CB CD34ⴙ
Plating efficiency, %
30.0
78.6*
Aggregate cell genotype
Uniform pattern
Frequent mixed nucleotide signal†
Heterogeneity of CD34ⴙ clones, %
Total rate
24.9*
1.6
Unique pattern
7.5*
1.2
Substitution (no.)
10.5 (64 of 611)*
0.0 (0 of 580)
Poly C tract (no.)†
11.9 (73 of 611)*
1.2 (7 of 580)
Length heteroplasmy at 303 to 315
10.8 (66 of 611)*
0.0 (0 of 580)
Length heteroplasmy at 16184 to 16193
1.1 (7 of 611)
0.5 (3 of 580)
Nucleotide change
0.0 (0 of 611)
0.7 (4 of 580)
2.5 (15 of 611)
0.4 (2 of 580)
Substitution plus poly C tract and others (no.)
Data are expressed as mutations per total assayed CD34⫹ clones.
*Statistically significant difference (P ⬍ .05) between 2 groups.
†Homopolymeric C tracts at nucleotides position at 303 to 315 and 16184 to 16193 in HV2 and HV1, respectively.
potentially affecting the efficiency of mitochondrial oxidative phosphorylation.16 Control region mtDNA mutations and variants therefore have
been hypothesized to impact longevity, climatic adaptation, and some
diseases such as type II diabetes16,17 and human tumors.18 One possible
speculation based on our results is that age-related control region
mutations in CD34⫹ clones could lead to derangement of marrow
function in older individuals. Empirically, coding regions might be
experimentally correlated with respiratory function in marrow cell populations. However, cells appear to have remarkable tolerance for decreased
mitochondrial activity,19 and the likely presence of mutations elsewhere in
the mtDNA genome might also affect function. Indeed, functional
measurements of oxidative phosphorylation in myelodysplastic bone
marrow samples have suggested abnormal function.20 Nevertheless, a
likely alternative possibility is that accumulated mtDNA mutations, while
advantageous to the DNA species by, for example, favoring replicative
efficiency, are neutral as related to overall cell or organ function.
Our data suggest age-related accumulation of somatic mtDNA
mutations in normal human hematopoietic tissue. As in other organs, it
appears probable that reactive oxygen species, generated in the mitochondria, lead to oxidative damage of the mtDNA.9 Mutated mtDNA copies
almost always would be underrepresented in aggregate samples due to
their small number and only detectable by analysis of individual cells
undergoing clonal expansion—in our experiments after in vitro cell
culture but in vivo after malignant transformation. Our data are
consistent with some recent publications; for example, acquisition of a
specific, easily detected substitution that leads to a well-described
polymorphism (C150T) was far more common in the leukocytes of very
aged individuals and showed an apparent tendency to increase with
age.21 Single cell analysis of human cardiomyocytes and buccal
epithelial cells also revealed a remarkably high frequency of homoplasmic expansion of mtDNA point mutations and deletions in persons over
50 years of age.22 Multiple mtDNA genotypes were present in closely
proximate hair follicles; while individual cells were not analyzed, the
interpretation of the investigators was that mtDNA mutations occur with
aging and resolve to homoplasmy within cells.23,24 Primary tissue and
cell lines of colon tumor origin also show a high frequency of mtDNA
mutations, presumably due to fixation of established homoplasmic
nucleotide changes.25 More generally, our results are concordant with
observations of surprisingly rapid divergence and resolution to homoplasmy of mtDNA sequence among inbred Holstein cows26 and in
carefully studied human kindreds.27,28
The molecular mechanisms responsible for expansion of mutant
mtDNA molecules to homoplasmy within a cell are obscure. mtDNA
properties of polyploidy and relaxed replication and the need to regulate
intracellular mtDNA copy number are believed to contribute to random
genetic drift13 and ultimately also to the clonal expansion of mtDNA
forms over extended periods of time.29 This physiology has been more
amenable to computer simulation than to empirical experimentation.7
Also unclear is the regulation of replicative fidelity in the homopolymeric
C tract, in which we localized much cell-to-cell heterogeneity and which
has been described by others as a mutational “hotspot”.30,31 The relative
proportion of variable length poly C tracts appears to be actively
maintained during cell division despite evidence of random mtDNA
segregation, suggesting de novo regeneration of specific pattern following
cell division by an as yet unknown molecular mechanism.32 Poly C and
other hypervariable region variants have been associated with several
common human diseases such as diabetes mellitus,17 low birth weight,33
and dilated cardiomyopathy.34 The pathophysiology of mtDNA disease is
confusing, and alterations in mtDNA may have tissue-specific consequences. For BM, accumulation of mtDNA mutations may relate to
normal aging of the hematopoietic compartment, and specific mtDNA
Table 6. Distribution of mtDNA heterogeneity from adult BM CD34ⴙ clones according to each grade and culture media
Grade
1
2
3
4
Subtotal no.
ⴙ
ⴚ
ⴙ
ⴚ
ⴙ
ⴚ
ⴙ
ⴚ
ⴙ
ⴚ
Total no.
1
3
3
1
1
5
7
10
0
19
11
30
2
2
0
1
0
0
0
0
0
3
0
3
3
2
3
3
3
3
4
5
1
13
11
24
4
1
1
1
3
4
2
0
4
6
10
16
5
6
6
6
6
10
7
10
1
32
20
52
6
0
1
3
5
2
0
1
1
6
7
13
Donor
Subtotal no.
14
14
15
18
24
20
26
7
79
59
138
Assayed clone no.
81
77
68
71
77
75
121
41
347
264
611
Proportion, %*
17.3
18.2
22.1
25.4
31.2
26.7
21.5
17.1
22.8
22.3
*No significant statistical differences were found between culture media with and without G-CSF and each grade. Abbreviations (⫹, –) are explained in the footnote to Table 1.
22.6
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BLOOD, 15 JANUARY 2004 䡠 VOLUME 103, NUMBER 2
lesions may affect the phenotype of an expanded population of abnormal
cells in clonal hematologic diseases such as leukemia or myelodysplasia.
Heterogeneity of mtDNA sequence in normal adult CD34⫹ cells
may have several implications. The possible relationship of mtDNA
sequence changes to hematopoietic aging has been discussed above. For
blood samples and, if our results apply to other tissues, in general
caution may be needed in forensic identifications and anthropologic
studies that depend primarily on the considerable sequence variation
found in the 2 hypervariable segments (HV1 and HV2). For the study of
hematopoiesis, mtDNA variant sequences may provide a natural genetic
“marker” for the determination of the contribution of individual stem
cells to blood cell production. Fixed mtDNA changes in leukemic cells
might provide a simple and nearly universally applicable method of
monitoring minimal residual disease. Finally, mtDNAsequence variability might be adapted to the measurement of the mammalian mtDNA
mutation rate. CD34⫹ cells in blood and marrow have virtually identical
properties, so blood sampling rather than marrow aspiration could be
mtDNA HETEROGENEITY IN SINGLE CD34⫹ CLONES
561
utilized to determine the mutational history of an individual, following
exposure to endogenous reactive oxygen species or environmental
mutagens. Because mitochondria are believed to have derived from
bacteria, mtDNA mutations in individual cells may allow development
of an “intracellular” in vivo Ames test for mutations.
Acknowledgments
This paper is a contribution of the US National Institutes of Health (NIH)
and the National Institute of Standards and Technology (NIST) and is not
subject to copyright. Certain commercial equipment, instruments, materials, or companies are identified in this paper to specify the experimental
procedure. Such identification does not imply recommendation or endorsement by NIH and NIST, nor does it imply that the materials or equipment
identified are the best available for this purpose.
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2004 103: 553-561
doi:10.1182/blood-2003-05-1724 originally published online
September 22, 2003
Marked mitochondrial DNA sequence heterogeneity in single CD34+ cell
clones from normal adult bone marrow
Myung Geun Shin, Sachiko Kajigaya, J. Philip McCoy, Jr, Barbara C. Levin and Neal S. Young
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