Somatic Progenitor Cell Vulnerability
to Mitochondrial DNA Mutagenesis
Underlies Progeroid Phenotypes in Polg Mutator Mice
Kati J. Ahlqvist,1Riikka H. Ha ¨ma ¨la ¨inen,1Shuichi Yatsuga,1Marko Uutela,1Mu ¨gen Terzioglu,6,7Alexandra Go ¨tz,1
Saara Forsstro ¨m,1Petri Salven,2Alexandre Angers-Loustau,3Outi H.Kopra,4,8Henna Tyynismaa,1Nils-Go ¨ranLarsson,6,7
Kirmo Wartiovaara,3,5Tomas Prolla,9Aleksandra Trifunovic,6,10and Anu Suomalainen1,11,*
1Research Programs Unit, Molecular Neurology, Biomedicum-Helsinki
2Research Programs Unit, Molecular Cancer Biology, Biomedicum-Helsinki
3Developmental Biology, Institute of Biomedicine
4Haartman Institute, Department of Medical Genetics and Research Programs Unit, Molecular Medicine, and Neuroscience Center
5Institute of Biotechnology
University of Helsinki, 00290 Helsinki, Finland
6Department of Laboratory Medicine, Karolinska Institutet, S-14186 Stockholm, Sweden
7Max Planck Institute for Biology of Aging, 50931 Cologne, Germany
8Folkha ¨lsan Institute of Genetics, 00290 Helsinki, Finland
9University of Wisconsin, Department of Genetics, Madison, WI 53706, USA
10Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, Cologne University, D-50674 Cologne, Germany
11Helsinki University Central Hospital, Department of Neurology, 00290 Helsinki, Finland
Somatic stem cell (SSC) dysfunction is typical for
different progeroid phenotypes in micewith genomic
defective Polg exonuclease activity also leads to
progeroid symptoms, by an unknown mechanism.
We found that Polg-Mutator mice had neural (NSC)
and hematopoietic progenitor (HPC) dysfunction
already from embryogenesis. NSC self-renewal was
decreased in vitro, and quiescent NSC amounts
were reduced in vivo. HPCs showed abnormal line-
age differentiation leading to anemia and lymphope-
nia. N-acetyl-L-cysteine treatment rescued both
NSC and HPC abnormalities, suggesting that subtle
ROS/redox changes, induced by mtDNA mutagen-
esis, modulate SSC function. Our results show that
mtDNA mutagenesis affected SSC function early but
manifested as respiratory chain deficiency in nondi-
deletions in postmitotic cells and no progeria, had
normal SSCs. We propose that SSC compartment is
sensitive to mtDNA mutagenesis, and that mitochon-
drial dysfunction in SSCs can underlie progeroid
Somatic stem cell (SSC) dysfunction has been proposed to lead
to decreased ability for tissue regeneration and aging (Schles-
singer and Van Zant, 2001; Sharpless and DePinho, 2007). This
hypothesis was supported by mouse models with genomic
DNA repair defects, which showed premature aging-like pheno-
types and severe SSC dysfunction (Frappart et al., 2005; Ito
et al., 2004; Narasimhaiah et al., 2005; Nijnik et al., 2007; Rossi
et al., 2007). Mice with Ku70 and Ku80 inactivation, leading to
defective nonhomologous DNA end-joining, had disrupted lym-
phopoiesis (Gu et al., 1997) as well as decreased amounts
of neural and hematopoietic progenitors (Narasimhaiah et al.,
2005; Rossi et al., 2007). A mouse model for trichothiodystrophy
(TTD), with a nuclear DNA helicase (xpd) mutation and defective
nucleotide excision repair, showed a premature aging-like phe-
etic progenitors (Rossi et al., 2007). Furthermore, mice with
ataxia-teleangiectasia (Atm) inactivation had reduced cell-cycle
checkpoint activity in response to DNA damage and telomeric
instability, resulting in infertility and self-renewal defect of hema-
topoietic stem cells (HSCs) (Barlow et al., 1996; Ito et al., 2004).
The progressive bone marrow failure of these mice was associ-
ated with elevated reactive oxygen species (ROS), and the HSC
defect could be reversed by treating the animals with n-acetyl-L-
cysteine (NAC), a compound with antioxidant capacity and an
effect on redox balance (Ito et al., 2004). Double inactivation of
ation and self-renewal defect of adult neural stem cells (NSCs)
in vitro, as well as decreased amount of dopaminergic neurons
in substantia nigra (Wong et al., 2003). These reports indicate
that SSCs are sensitive to defects of genomic DNA repair, and
suggest a role for ROS and/or redox status in regulating SSC
quiescence and proliferation. They also strongly suggest that
SSC dysfunction is intimately connected with premature aging-
like symptoms in mice.
in progeroid phenotype in mice. ‘‘Mutator’’ mice show exten-
sive mtDNA mutagenesis, with point mutations and complex
100 Cell Metabolism 15, 100–109, January 4, 2012 ª2012 Elsevier Inc.
rearrangements, because of an inactivated exonuclease func-
tion of the mitochondrial replicative DNA polymerase gamma
(POLG) (Ameur et al., 2011; Kujoth et al., 2005; Trifunovic
et al., 2004; Williams et al., 2010). Their phenotype mimics
premature aging, starting from 6–8 months of age, with progres-
sive hair graying, alopecia, osteoporosis, general wasting, and
reduced fertility (Kujoth et al., 2005; Trifunovic et al., 2004). Their
life span is limited to 13–15 months because of severe anemia,
with age-dependent decline in erythro- and lymphopoiesis,
recently suggested to be due to HSC dysfunction (Chen et al.,
2009; Norddahl et al., 2011). POLG forms the minimal mtDNA
replisome together with the mitochondrial single-stranded DNA
binding protein and a replicative helicase, Twinkle (Korhonen
et al., 2004; Spelbrink et al., 2001). If dominant mutant Twinkle
is overexpressed in mice, large-scale mtDNA deletions accumu-
late in postmitotic tissues (hence the ‘‘Deletor’’ mice), leading to
progressive late-onset mitochondrial myopathy at 12 months of
age and respiratory chain (RC)-deficient neurons, but normal life
span without progeroid features (Tyynismaa et al., 2005).
We asked whether SSC dysfunction could contribute to the
mtDNA mutagenesis-linked premature aging, and utilized the
two mouse models with mtDNA maintenance defects, Mutator
and Deletor, of which only the former showed a premature aging
phenotype. We report here NSC and HSC dysfunction in murine
Old Mutator Neurons and Skeletal Muscle Show Mild
Respiratory Chain Deficiency
To characterize the consequences of high mtDNA mutation
load in postmitotic cells, we analyzed the activity and presence
of RC enzymes of Mutator mice at the time when they already
manifest progeroid phenotype, with hair loss, kyphosis, and
severe anemia (13–14 months, Figures 1A and 1B). Histochem-
ical enzyme assay of cytochrome c oxidase (COX, partially en-
coded by mtDNA) and succinate dehydrogenase (SDH, nuclear
encoded) showed no COX-negative, SDH-positive neurons in
the hippocampus or dentate nucleus (Figures 1C and 1D), or
any area with large neurons, including the cortex and cere-
bellum. However, as previously described (Tyynismaa et al.,
2005), Deletor mice at the age of 18 months showed occa-
sional COX-negative neurons in all areas harboring large neu-
rons, including hippocampus and dentate nucleus (Figures 1C
and 1D). Immunohistochemistry for RC complexes I (Figure 1F
and see Figure S1C available online) and II and IV (Figure S1C)
showed high content of these complexes, with only occasional
CI-low Purkinje cells in cerebellum, and were similar to WT in
the cortex even at 46 weeks of age (Figures S1A and S1B). By
western blot, Mutator brains showed a tendency to reduced
amounts of complex I (80% of WT) and COX (72% of WT) (Fig-
ure 1H). No signs of gliosis were seen in the Mutator brain
even at the age of 46 weeks, in SVZ, hippocampus, cortex,
and striatum (Figures S1F), and the number of neurons was
similar in Mutators and WT littermates, when counted from
hippocampal regions CA1 and dentate gyrus (Figures S1G
and S1H). Skeletal muscle of Mutator showed less than 1% of
COX-negative fibers, whereas Deletors showed ?5% of total
muscle fibers to be COX negative (Figure 1G), and the heart
showed patches of COX-negative cardiomyocytes (data not
shown). The late onset of the Mutator mitochondrial myopathy
was further supported by the increase of FGF21 cytokine in
serum of the Mutators (Figure 1I), which we recently have shown
to be secreted from COX-negative muscle fibers in Deletor mice
and to correlate with the degree of RC deficiency (Tyynismaa
et al., 2010). Contrasting the otherwise good COX activity
in CNS, the neurogenic region of subventricular zone (SVZ)
(Figures 2A and 2B), and the metabolically active choroid plexus
(Figure 1E) had high numbers of COX-negative cells in the Muta-
tors. SVZ was normal in Deletors (Figure 2B), but choroid plexus
showed occasional COX-negative cells (Figure 1E). Apoptosis
was not induced in the SVZ of 40- to 46-week-old Mutators
These results indicate that even at the terminal phase of the
severe progeroid phenotype of mtDNA Mutator mice, their
neurons and muscle cells can maintain a well-preserved RC.
Mutators Show Decreased Neural Progenitors
in the Adult Brains
As the adult Mutator brains showed RC deficiency in SVZ,
we asked whether the neural progenitors were affected. We
nestin positivity of adult Mutator brain (Figures 2C and 2D) and
compared it to Deletor and WT mice. Statistically significant
decrease in nestin-positive cells was seen in >40-week-old
Mutator SVZ when compared to WT littermates, whereas Dele-
tors showed WT-like staining. Proliferative CDC47-positive cells
in Mutator (Figure 2E) and in Deletor SVZ were similar to WT in all
ages. Since the SVZ region is feeding neural progenitors to the
olfactory bulb throughout the life, we analyzed the number of
olfactory bulb periglomerular cells. The number of calbindin-
compared to WT littermates in different time points (Figure 2F).
These results show that the amount of nestin-positive NSCs is
reduced in the adult Mutator SVZ, but during the restricted life-
Our results show that even at an advanced stage of premature
aging syndrome, the central nervous system shows a minimal
phenotype, except for partial loss of quiescent nestin-positive
SVZ progenitors. Three subtypes of NSCs have been identified
from the SVZ region (Miller and Gauthier-Fisher, 2009; Suh
et al., 2009). Type B cells, resembling radial glial cells that serve
as NSCs during development, are slowly or infrequently dividing,
whereas type C cells are actively proliferating. Type B cells
express nestin and GFAP, whereas type C cells are both
nestin and GFAP negative. Our results strongly suggest that
the quiescent type B cells were decreased in the old Mutator
brains. However, in Deletors at the age when neurons were
clearly affected with RC deficiency (18 months), no indication
of abnormal SVZ was seen.
mtDNA Mutagenesis Leads to Decreased Self-Renewal
Capacity of Neural Stem Cells In Vitro, Attenuated with
To obtain understanding of the Mutator SSC phenotype, we
cultured NSCs from E11.5–E15.5 Mutator embryos as freely
floating neurospheres. Spheres from E15.5 Mutator embryos
mtDNA Mutagenesis Affects Somatic Stem Cells
Cell Metabolism 15, 100–109, January 4, 2012 ª2012 Elsevier Inc. 101
compared to the WT spheres (Figure 3A). At the protein level,
the increased mtDNA mutations resulted in slight reduction of
RC complexes I and IV in NSCs (Figures 3B and 3C), but com-
plexes II and III did not differ from WT when quantified against
significantly highermtDNA point mutationload
Figure 1. Old Mutator Brain and Skeletal Muscle
Show Mild Respiratory Chain Deficiency
(A) At 13 months of age, Mutator mice manifest early-
onset progeria with alopecia and kyphosis as well as (B)
terminal-stage anemia. Hb values shown as mean ± SD.
WT (n = 2), 119.5 ± 3.5; Mutator (n = 3), 52.67 ± 24.1;
Deletor (n = 12), 114.1 ± 12.7. (C–G) Samples of Mutator,
13 months; WT, 18 months; Deletor, 18 months. Cry-
osections showing cytochrome c oxidase (COX, brown
staining) and succinate dehydrogenase (SDH, blue stain-
activity assay from (C) hippocampus (scale bar, 500 mm;
insets from CA2 region 10 mm); (D) cerebellar dentate
nucleus (scale bar, 20 mm). No COX-negative, SDH+cells
were found in Mutator hippocampus or cerebellum at the
age of terminal manifestation, whereas Deletors at the age
of 18 months showed occasional COX-negative, SDH+
cells (blue) in all areas with large neurons. (E) In metabol-
ically active choroid plexus, Mutators showed high
numbers of COX-negative cells (blue), similar to Deletors,
but also WT 18-month-old mice had occasional COX-
negative cells (scale bars, 10 mm). (F) RC complex I
immunohistochemical staining of Purkinje cell layer.
Mutators show occasional CI-low cells, but typically ex-
cellent staining of CI (brown; counterstained with hema-
toxylin), whereas Deletors show high variability of CI
staining (scale bar, 40 mm). (G) COX-SDH activity assay of
frozen sections of skeletal muscle. Less than 1% of
Mutator skeletal muscle fibers were COX negative,
whereas in Deletors 5% of total muscle fibers were COX
negative (scale bar, 20 mm). (H) Mitochondrial extracts
from Mutator and WT brain at 39 weeks of age were
analyzed by western blot, and Mutator brain showed
IV (72% of WT). Complexes I, III, and IV were normalized
against nuclear-encoded complex II, and results are
two separate protein extracts per animal, WT n = 1 and
Mutators only at 37–40 weeks of age (24–25 w; p = 0.559,
37–40 w; p = 0.026). See also Figure S1.
nuclear-encoded mitochondrial outer mem-
brane protein, porin (Figure 3C). Also the cyto-
chrome c oxidase activity was normal (COX
activity/mitochondrial matrix enzyme, citrate
synthase [CS] activity, WT NSCs [n = 4 lines]
0.66 ± 0.31; Mutator NSCs [n = 3] 0.56 ± 0.06).
We next examined whether mtDNA mutations
affected the self-renewal capacity of NSCs
in vitro. The primary proliferation characteristics
of early passage (<6) NSCs, measured by in-
corporation of BrdU and by flow cytometry,
showed that mtDNA mutations did not affect
the proliferative activity of NSCs (Figure 3D).
To test the NSCs’ ability to self-renew, cultures
were diluted to clonal density, and the single-
cell ability to produce new neurospheres was monitored. Muta-
tor NSCs formed ?3-fold less spheres when compared to WT
NSCs (Figure 3E).
affected by increasing redox buffer capacity and antioxidant
mtDNA Mutagenesis Affects Somatic Stem Cells
102 Cell Metabolism 15, 100–109, January 4, 2012 ª2012 Elsevier Inc.
action. Heterozygous females were fed with NAC throughout the
pregnancy and NSCs were extracted from E14.5 embryos, and
cultures were supplemented with NAC. Self-renewal ability of
treated and nontreated NSCs was monitored and showed that
NAC treatment restored the self-renewal ability of Mutator
NSCs but had no significant affect on the WT NSCs (Figure 3E).
not during embryonal life, it had no effect on self-renewal ability
of any NSCs (data not shown). During long-term culture, Mutator
NSCs also showed growth restriction: most lines were unable to
grow more than 10 passages, whereas WT neurospheres con-
tinued growth unaffected after 50 passages (Figure 3F). NAC
NSCs but instead caused high variability to WT NSCs’ growth
properties (Figure 3G). Next we examined NSCs’ multipotency
by inducing differentiation of single-cell-derived neurospheres
to progenitor lines. MutatorNSCs were able to produce morpho-
logically similar neurons and astrocytes as WT (Figure 3H). Our
data show that Mutator NSCs have a WT-like capacity to differ-
entiate to different cell types, but reduced self-renewal capacity,
which was attenuated by NAC supplementation. However, the
growth defect of long-term cultures was not affected by NAC.
These results suggest that the mechanism of NSC self-renewal
defect involves ROS/redox status.
Mutators Show Abnormal Fetal Erythropoiesis
and Disrupted Hematopoietic Progenitor Differentiation
in the Adult Bone Marrow
The Mutator NSC phenotype prompted us to ask whether other
stem/progenitor cell compartments were affected in the devel-
oping embryo. Adult Mutator mice have been shown to develop
severe progressive anemia after 6 months of age (Figure 1B)
(Chen et al., 2009; Trifunovic et al., 2004) with progressive
dysfunction of bone marrow HSCs (Chen et al., 2009; Norddahl
et al., 2011). We could replicate the previous findings from adult
bone marrow: despite their severe anemia, the adult Mutator
Figure 2. Adult Mutator Brains Show COX-Negative Cells and Decreased Levels of Nestin-Positive NSCs in the SVZ, but the Amount of
Proliferative Cells in the SVZ, as well as Olfactory Bulb Interneurons, is Wild-Type Like
(A) Midsagittal view of adult mouse brains showing the areas of neurogenesis in red. Newly formed neural progenitors migrate from SVZ through rostral migratory
stream (RMS) to olfactory bulb (OB), where they give rise to interneurons maintaining the OB function.
(B) The SVZ (arrows) had high numbers of COX-negative cells in Mutators, visualized by COX-SDH activity from cryosections (Scale bar, 10 mm), but not in the
Deletors or WT mice.
(C) Old Mutator brains at 14 months show decreased number of nestin-positive NSCs in SVZ region, whereas in Deletors at 18 months the NSC numbers did not
differ from WT mice.
(D) The relative optical density of the nestin signal was analyzed from SVZ region (600–1,200 cells per sample) and normalized against WT sample. Shown as
mean ±SD, ***p < 0.0001; animals per genotype, n = 2; scale bar, 20 mm.
positive cells shown as percentage of total cells, mean ±SD. Animals per genotype: 14 weeks, n = 2;25 weeks, n = 3;>40 weeks, n = 2.Shown is a representative
picture of CDC47 immunostaining, proliferating cells seen in brown, from 46-week-old Mutator and WT brains. Scale bar, 20 mm.
(F)Thenumberof olfactorybulbperiglomerularinterneurons,ofwhichasubsetis calbindinpositive,wassimilarto WT inoldMutatorbrains.Threehundredtofour
mtDNA Mutagenesis Affects Somatic Stem Cells
Cell Metabolism 15, 100–109, January 4, 2012 ª2012 Elsevier Inc. 103
bone marrow showed well-maintained total cellularity (results
shown as mean ± SD, WT [n = 2] 9.6 3 106± 1.1 3 106and
Mutator [n = 2] 7.2 3 106± 2.3 3 106). Even at 40 weeks, all
cell populations expressing the erythroid markers typical for
each maturation stage were present, but the erythroid FACS
profiles were clearly abnormal, with lower mean intensity of
Ter119 signal compared to the WT bone marrow (Figures S2A
and S2B). Both the myeloid and B-lymphoid cell numbers were
decreased in the 40-week-old adult Mutator bone marrow
The end stage severe HSC phenotype suggested that HSCs
could be affected already in early phase. We investigated
whether the hematopoietic system and erythropoiesis were
established during embryonic development. At E13–E15.5, the
major site for mouse hematopoiesis is the liver (Ema and Nakau-
chi, 2000). We detected a decrease in the total amount of cells
in Mutator fetal liver extracts compared to WT littermates (Fig-
ure 4A). Based on FACS analysis, the major hematopoietic line-
ages, myeloid and lymphoid, were established normally in
of different erythroid precursors differed from WT already in the
early embryo: basophilic erythroblasts (CD71+,Ter119+ popula-
tion) progressively decreased in the Mutators (Figure 4B), and
the most mature erythroid progenitors (Ter119+,CD71low)
Figure 3. Cultured Mutator NSCs Accumulate mtDNA Point Mutations and Show Decreased Self-Renewal Capacity and Growth Defect in
Long-Term Culture, which Can Be Attenuated by NAC Supplementation
(A) Cultured Mutator (n = 3) NSCs from E15.5 embryos showed increase in point mutation load in the cytochrome B gene of mtDNA compared to WT (n = 4) NSCs
(Mutator 13.7 mutations/10 kb; WT 0.2 mutations/10 kb).
(B) Mutator NSCs (n = 3) showed slight reduction of RC complexes I and IV analyzed by western blot.
(C) Mutator NSCs (n = 3) RC protein levels were compared to WT NSCs (n = 4), and the amount of RC subunits was normalized to nuclear-encoded mitochondrial
(D) Mutator NSCs (n = 4) showed BrdU incorporation similar to that of WT (n = 8) NSCs, indicating unaffected proliferation capability in flow cytometric analysis
when 120,000–240,000 cells per genotype were analyzed. Analysis was performed with low-passage NSCs (p < 6).
(E) Mutator NSCs (n = 13) produced significantly less neurospheres than did WT (n = 18; ***p = < 0.0001) or Deletor (n = 6;***p = 0.0005) NSCs in clonal expansion
analysis, indicating reduced self-renewal capacity. Mutator NSCs with NAC (n = 11) showed improved self-renewal capacity (***p < 0.0001), while the treatment
had no significant effect on the WT NSCs (n = 7; p = 0.321). Altogether 10,500–27,000 cells per genotype were analyzed. Self-renewing cells are shown as
a percentage of total cells (mean ±SD): Mutator, 6.5% ± 3.4%, WT, 19.9% ± 7.7%, and Deletor, 16.5% ± 2.8%; Mutator with NAC, 15.8% ± 5.3%, and WT with
NAC, 23.3% ± 6.5%.
(F) Mutator NSCs (n = 3) showed growth restriction in continuous long-term culture compared to WT NSCs (n = 5), and the difference between WT and Mutator
lines became statistically significant by the ninth week of culture.
(G) The growth restriction of Mutator NSCs was partially attenuated by NAC supplementation: Mutator NSCs with NAC (n = 6) grew slightly better compared to
Mutator NSCs but could not reach the same passage levels as WT or WT-NAC (n = 6) lines.
(H) Cultured Mutator NSCs were able to differentiate into both neuronal (Tuj-1, green) and glial (GFAP, red) cells when induced to differentiate with 2% FBS.
Hoechst (blue) was used to stain nuclei.
mtDNA Mutagenesis Affects Somatic Stem Cells
104 Cell Metabolism 15, 100–109, January 4, 2012 ª2012 Elsevier Inc.
increased in the fetal liver at E15.5 (Figure 4C). The proerythro-
blast population (CD71+) was similar to WT (results shown as
mean ± SD, E13-E13.5: WT [n = 7] 9.1% ± 2.6% of total cells;
Mutator [n = 3] 8.1% ± 0.5%; E15.5: WT [n = 15] 8.4% ± 2.8%;
WT NAC [n = 12] 7.5% ± 1.8%, Mutator [n = 7] 8.9% ± 1.5%;
Mutator NAC [n = 10] 8.0% ± 0.9%).
We next tested whether we could affect the fetal hemato-
poietic phenotype with NAC supplementation. Heterozygous
females were fed with NAC throughout the pregnancy, and
hematopoietic cells were extracted from the liver of E15.5
embryos and subjected to FACS analysis immediately after
extraction. The total amount of cells in the fetal liver of Mutators
was restored to WT levels by NAC supplementation (Figure 4A).
Also the proportions of different erythroid progenitor populations
were normalized to WT level in the Mutator embryos treated with
NAC (Figures 4B and 4C). We then analyzed the frequency of
CD11b-positive myeloid and B220-positive B-lymphoid progen-
itors in fetal liver and found a progressive increase in the amount
of B-lymphoid cells in the Mutators compared to WT littermates
(Figure 4D). However, NAC-treated Mutator E15.5 embryos
ure 4D). These results show that, similar to the NCS defect, the
fetal hematopoietic phenotype can be ameliorated by NAC
To analyze the function of fetal HPCs, we analyzed their ability
to produce hematopoietic colonies when plated on methylcellu-
lose. Mutator fetal liver HPCs were able to produce mixed mye-
loerythroid colonies (CFU-GEMM), whereas erythroid (BFU-E)
and granulocyte-macrophage colonies (CFU-GM) were mod-
estly increased in number (Figure S2E). Forty-week-old Mutator
granulocyte-macrophage colonies than did WT (Figure S2F).
These results show that different lineages of the hematopoietic
system appear to be established during embryogenesis,
although distorted erythroid and lymphoid lineage differentiation
mentation was able to bring the frequencies of the erythroid and
lymphoid cell populations in Mutator fetal liver to WT level.
Deletors Show No mtDNA Deletion Formation in NSCs,
and Have Normal NSC Characteristics and
Deletor mice, which accumulate mtDNA deletions and have RC
deficiency in their postmitotic tissues (Figures 1B–1G) (Tyynis-
maa et al., 2005), displayed neither increased mtDNA point
mutation load (1.1/10 kb in Deletors, 0.7/10 kb in WT littermates)
nor mtDNA deletions (data not shown) in their NSCs, and were
also able to self-renew (Figure 3E) and differentiate normally
when compared to WT littermates. The mutant transgene was
expressed in NSCs 2-fold compared to the endogenous gene,
which is similar to the level that causes mtDNA deletions in the
Deletor skeletal muscle, brain, and heart. Hematopoiesis was
normal in the Deletors: their erythroid, lymphoid, and myeloid
progenitor amounts were similar to WT both in fetal liver and in
adult bone marrow from animals of 110 weeks of age (Figures
nance defect affects SSC function only when mtDNA mutation
load is increased in the stem/progenitor cells themselves.
We report here that the mtDNA Mutator mice with premature
aging-like syndrome have an embryonal-onset progressive
dysfunction of neural and hematopoietic progenitor cells,
leading to reduction of quiescent neural progenitors in the SVZ
and to severe anemia and lymphopenia in the adults. Previously,
Figure 4. Mutators Show Abnormal Eryth-
ropoiesis and Lymphopoiesis
during Fetal Development, Attenuated by
(A) Mutator fetal liver extracts showed slightly
decreased amount of total cells compared to WT
littermates at E15–E15.5 (WT, n = 15; and Mutator,
n = 7; p = 0.099). NAC supplementation during the
fetal development increased the total fetal cell
count in both WT (n = 12) and Mutator (n = 10)
embryos, bringing the Mutator cell number to the
decreased during the embryogenesis of Mutator
mice, whereas (C) the amount of more ma-
ture orthochromatophilic erythroblasts (Ter119+,
CD71low) increased. NAC supplementation during
the fetal development restored both cell pop-
ulations in Mutator fetal liver to the WT level
(E13–E13.5 WT, n = 9; Mutator, n = 8; E14–E14.5
WT, n = 3; Mutator, n = 4; E15.5 WT, n = 15; WT
NAC, n = 12; Mutator, n = 7; and Mutator NAC,
n = 10). (D) The amount of B220-positive
B-lymphoid cells increased in Mutator fetal liver,
reaching statistical significance at E15.5 (p =
0.0092), while NAC supplementation during fetal
development was able to normalize the frequency of B220-positive cells (E13–E13.5 WT, n = 9; Mutator, n = 5; E14–E14.5 WT, n = 3; Mutator, n = 8; E15.5 WT,
n = 15; WT NAC, n = 12; Mutator, n = 7; and Mutator NAC, n = 10). Graphs in (B)–(D) show the percentage of positive cells from total fetal liver cells (mean ±SD).
mtDNA Mutagenesis Affects Somatic Stem Cells
Cell Metabolism 15, 100–109, January 4, 2012 ª2012 Elsevier Inc. 105
progeroid mice with dysfunctional nuclear DNA repair have been
reported to have similar defects. The NSCs of prematurely aging
Atm?/?Terc?/?mice showed reduced self-renewal capacity
in vitro and decreased amount of proliferating cells in adult
SVZ region in vivo (Wong et al., 2003). Ku70?/?, Ku80?/?, and
TTD mice showed a decreased amount of hematopoietic pro-
genitors in their bone marrow and defective repopulation
activity of HSCs (Gu et al., 1997; Rossi et al., 2007). Similar to
mice showed progressive anemia and
decreased amount of granulocyte-macrophage and lymphoid
progenitors in their bonemarrow (Ito etal., 2004).The close simi-
larities between the stem cell properties of other premature
aging models and Mutators emphasize the importance of both
nuclear and mtDNA integrity in tissue progenitor homeostasis
Parallel to their severe SSC phenotype, the Mutator mice
developed modest late-onset signs of mitochondrial RC defi-
consequence in mtDNA Mutator, the surprisingly late and mild
nature of RC defect indicates that postmitotic tissues can resist
random mtDNA mutagenesis well. We compared the findings in
the Mutators to another mouse model with mtDNA maintenance
defect, the Deletors. The latter accumulate large-scale mtDNA
deletions in postmitotic tissues, leading to late-onset RC defi-
ciency, mimicking the muscle and brain phenotype of Mutators.
However, the Deletors have normal SSCs and no premature
aging phenotype. These findings support a crucial role of SSC
dysfunction in generating the progeroid phenotype of Mutators.
Previously, Mutator brains have been reported to have
RC deficiency, based on changes in macroscopic COX-SDH
activity, with no data on protein complexes or histology on
neuronal level (Ross et al., 2010; Vermulst et al., 2008). We did
not find abnormal COX or SDH activities in our old Mutator
brains, but only a marginal reduction of complex I and COX
protein amounts by western and immunohistochemical anal-
yses. While COX-negative neurons could readily be found in
the Deletors, they did not exist in Mutators, suggesting that
neurons can compensate random mtDNA mutagenesis well.
This is supported by the finding that mice with postnatal disrup-
tion of mitochondrial transcription factor A in cortical and hippo-
campal neurons, leading to loss of mtDNA, survived for months
before developing RC dysfunction and neurodegeneration (Sor-
ensen et al., 2001). Based on these results, we propose that the
Mutator mice have two separate manifestations: (1) a progeroid
syndrome as a consequence of early-onset dysfunction of
SSC pool, and (2) a late-onset mild RC deficiency in brain,
skeletal muscle, and heart as a consequence of progressive
SSC maintenance is dependent on a fine balance of self-
renewal and differentiation, in the regulation of which physio-
logical ROS has recently been implicated: low concentrations
of ROS, which did not cause detectable DNA, protein, or lipid
damage, could shift quiescent SSCs toward proliferation and/
or differentiation (Hamanaka and Chandel, 2010; Le Belle
et al., 2011; Shao et al., 2011; Yoneyama et al., 2010). Interest-
ingly, the NSC and HPC defects of Mutator could be amelio-
rated by NAC treatment, suggesting involvement of aberrant
ROS signaling or redox status in Mutator somatic precursor
cell maintenance. No direct evidence of oxidative damage to
proteins, lipids, or nucleic acids was found in the tissues or
cells of Mutators (Kujoth et al., 2005; Trifunovic et al., 2005),
but their cardiomyopathy was attenuated by overexpression of
mitochondrial-targeted catalase, a ROS scavenger (Dai et al.,
2010). In Atm?/?and FoxO1/3/4L/Lmice, even a small increase
in ROS production in HSCs could affect their quiescence and
disrupt their ability to reconstitute their niches (Ito et al., 2004;
Tothova et al., 2007). Similar to Mutators, the SSC phenotypes
of Atm?/?and FoxO1/3/4L/Lmice could be complemented with
NAC. NAC is a thiol, which is readily deacetylated to form
L-cysteine, the rate-limiting amino acid for reduced glutathione
(GSH) synthesis. Therefore, NAC increases the amount of GSH
in cells, improving their redox buffering capacity, but also has
direct antioxidant effects (De Flora et al., 1995; Murphy et al.,
2011). We suggest that a subtle increase in mitochondrial O2?
and/or change in the redox status, as a consequence of mtDNA
their function to severely disrupt SSC homeostasis.
Our data of NSCs and HPCs suggest that the Mutators have
a widespread dysfunction of tissue progenitors. The Mutator
NSCs showed reduced self-renewal capacity in vitro and failure
to maintain proliferation in long-term culture. In adult brains, the
number of the quiescent nestin-positive type B neural progeni-
tors of SVZ was decreased, a logical consequence of disability
to self-renew. Considering the early-onset NSC defect in vitro
in the Mutators, the adult brain phenotype was surprisingly
modest, suggesting that the life span of Mutators may be too
short to allow full manifestation of late-onset neurodegeneration.
No increase of apoptosis in the brain was seen, even in the
neurogenic regions, which, however, showed RC deficiency. In
Mutator brains, the cells of choroid plexus and SVZ neurogenic
area showed high numbers of COX-negative cells. Choroid
plexus sometimes shows COX-negative cells even in old WT
mice,whichissuggested tobecaused byhigh metabolicactivity
of these cells. However, RC deficiency in potential neural pro-
genitors raises the question of whether these cells preferentially
accumulate mtDNA mutations and whether their mtDNA half-life
is short and turnover rapid, making them dependent on POLG
function. These questions require further attention in the future.
In the adult bone marrow, at the time when the mice suffered
from severe anemia, different erythroid progenitor populations
diate states of maturity, supporting previous results (Chen et al.,
2009; Norddahl et al., 2011). Furthermore, we show that Mutator
hematopoiesis is affected already during fetal development,
when different progenitor populations are all established, but
present in aberrant amounts. These data suggest that mtDNA
mutagenesis affects the quality of hematopoietic progenitors
more than quantity, possibly disturbing their normal quiescent
state, which has been suggested to be crucial for reconstitution
capacity and long-term sustenance of HSCs (Arai et al., 2004).
Although we found no signs of exhaustion of undifferentiated
progenitor cells in Mutator bone marrow, our results do suggest
deranged differentiation pattern and function of erythroid pro-
genitor populations, which may result in increased apoptosis
of hematopoietic cells, as previously suggested (Norddahl
mutagenesis also to affect lymphopoiesis, leading to lymphope-
nia in adults, supporting a previous report (Chen et al., 2009).
mtDNA Mutagenesis Affects Somatic Stem Cells
106 Cell Metabolism 15, 100–109, January 4, 2012 ª2012 Elsevier Inc.
Paradoxically, Mutator embryos showed increased B-lymphoid
cells in the fetal liver, whereas the adult bone marrow showed
a low number of B-lymphoid progenitors and decreased ability
to produce myeloerythroid colonies. These data further sug-
gest that disrupted proliferation and/or differentiation of the
hematopoietic progenitor cells underlies Mutator anemia and
POLG is part of the minimal replisome of mtDNA together with
Twinkle helicase (Korhonen et al., 2004). Because of the neces-
sity of these two proteins for mtDNA replication, their defects
could be assumed to affect tissues with high proliferative
activity, such as the skin, the gastrointestinal tract, or the blood.
However, in humans, recessive POLG and Twinkle mutations
result in progressive childhood, juvenile, or adult-onset neurode-
generative disorders, with muscle and brain RC deficiency, but
no anemia or lymphopenia, or no apparent signs of premature
aging (Suomalainen and Isohanni, 2010). Therefore, it is some-
what surprising that exonuclease deficiency of POLG does not
mimic human POLG disorders but affects the proliferative
tissues that have high demand for mtDNA replication, especially
the progenitor populations. This suggests that catalytic POLG
functions involved in mtDNA repair are important for the long-
lived brain mtDNAs (Weissman et al., 2007), whereas the proof-
reading POLG function and faithful mtDNA replication are
especially essential in proliferating progenitors. However, no
Mutator-like phenotype with extensive random mutagenesis of
mtDNA has been characterized in humans, and therefore com-
parison of Mutator results to human diseases should be done
Recently, endurance exercise was reported to ameliorate
the premature aging symptoms in Mutator mice (Safdar et al.,
2011). Exercise has been shown to affect the vitality of muscle
and NSCs (Blackmore et al., 2009; Shefer et al., 2010; Wu
et al., 2008) and also to promote hematopoiesis (Baker et al.,
2011). It remains to be studied whether increased SSC fitness
underlies the effect of exercise in Mutators.
We show here that accumulation of mtDNA mutations in
Mutator mice already leads to alterations in two distinct stem/
progenitor cell compartments during fetal development. RC
deficiency in postmitotic tissues is a late phenomenon in these
mice, starting to manifest at the time when anemia and lympho-
penia are restricting the life of these animals. Our results show
that the hematopoietic compartment is especially vulnerable to
mtDNA mutagenesis, whereas the nervous system is well pre-
served. Our results of NAC complementation suggest that the
mechanism by which mtDNA mutations affect the homeostasis
of tissue progenitors involves physiological ROS/redox signaling
in SSCs, repressing quiescence state. Our results strongly sug-
gest that mtDNA mutagenesis in somatic stem/progenitor cells
can contribute to aging-related phenotypes.
See also the Supplemental Experimental Procedures.
All animal experimentation was approved by the Ethical Review Board of
Finland. Mice with a knockin inactivating mutation (D257A) in the exonuclease
domain of DNA polymerase gamma, PolG (Mutator mice [Kujoth et al., 2005;
Trifunovic et al., 2005]), and mice ubiquitously overexpressing mouse Twinkle
cDNA with a dominant human disease mutation, leading to duplication of
13 amino acids (dup353–365) in the linker region of mitochondrial Twinkle
helicase (Deletor mice [Tyynismaa et al., 2005]), were used. We used two
Mutator mouse colonies: one in C57Bl6, originating from T.P., and these
mice were used in NSC and HSPC experiments and in western analysis of
the RC complexes from NSCs. Embryos for HSPC and NSC extraction, as
well as adult brains for immunohistology and histochemistry, were obtained
from Mutator colony established by A.T. and N.-G.L.
Analysis of the Respiratory Chain Complexes by Western Blot
Whole-cell protein extraction from NSCs, mitochondrial protein enrichment
from brains, SDS-PAGE, and immunodetection of RC complexes were per-
formed as previously described (Ylikallio et al., 2010). For protein detection,
we used monoclonal antibodies against the 39 kDa subunit of complex I,
core 2 or Rieske subunit of complex III, cox1p subunit of complex IV, 70 kDa
Ip subunit of complex II (Mitosciences) and porin (VDAC) (Calbiochem) as
a loading control. All samples were analyzed in duplicates.
Respiratory Chain Enzyme Activity Measurements and FGF21
The RC enzyme activity of complex IV (cytochrome c oxidase) and the enzyme
activity of CS were determined from whole-cell lysates of cultured NSCs as
previously described (Suomalainen et al., 1992). All samples were analyzed
as duplicates. FGF21 levels in serum of the mice were analyzed by ELISA,
with Quantikine mouse FGF-21 Immunoassay kit (R&D Systems, MF2100) as
described (Tyynismaa et al., 2010).
NSCs from Embryos
NSCs were extracted from the lateral ventricular wall of E11.5–E15.5 mouse
brain as previously described (Piltti et al., 2006). Neurospheres were cultured
in serum-free F12 medium (Sigma-Aldrich) supplemented with FGF2 and EGF
mtDNA Deletion and Point Mutation Analysis
DNA from low-passage (<10) neurospheres was extracted by standard
phenol-chloroform and ethanol precipitation method. MtDNA deletions were
analyzed as previously described (Tyynismaa et al., 2005). For point mutation
analysis, the CytB and control region areas of mtDNA were amplified as
described (Trifunovic et al., 2004) with a high-fidelity DNA polymerase (Phu-
sion, New England BioLabs). PCR products were cloned into ZeroBlunt
Topo vector (Invitrogen) and sequenced. Altogether, 30 kb of sequence was
analyzed from each genotype from both CytB and control region using
Sequencher software (Genecodes). Single mutations were counted once, to
avoid repeated calculations of clonal mutation events, but this strategy
ignored same-site mutations.
Analysis of NSC Self-Renewal Capacity
Analysis of NSC self-renewal capacity was performed as previously described
(Piltti et al., 2006).
BrdU Incorporation Assay
To determine the proliferation rate, neurospheres were incubated in 10 mM
BrdU (BD PharMingen), stained with anti-BrdU and fluorescent secondary
antibody, and analyzed using FACSAria Cell Sorter.
N-Acetyl L-Cysteine Supplementation
Mice were given 1 mg/ml of NAC (Sigma-Aldrich) in drinking water throughout
the pregnancy. NSCs were extracted from E14.5 embryos, and culture
medium for NSCs was supplemented with 100 mM NAC. HSCs were extracted
from E15.5 embryos and subjected to FACS analysis immediately after the
Differentiation Analysis of NSCs
Neurospheres originating from single cells were induced to differentiate using
2% FBS and poly-D-lysine-coated chamber slides; cells were stained with
anti-Tuj-1 and anti-GFAP, and presence of neurons and astrocytes was
mtDNA Mutagenesis Affects Somatic Stem Cells
Cell Metabolism 15, 100–109, January 4, 2012 ª2012 Elsevier Inc. 107
of 72 weeks were collected and standard hematoxylin and eosin (TissueTek)
protocol was used to determine the morphology from paraffin sections. RC
complex subunits were visualized using rabbit anti-CI ND-1 (1:500), mouse
anti-CI NDUFS3 (1:100), mouse anti-CII 70 kDa subunit (1:200), and mouse
anti-CIV II (1:20). Mouse anti-CDC47 (1:200), rabbit anti-cleaved caspase 3
(1:100), mouse anti-nestin (1:200), and mouse anti-myelin basic protein
(1:200) were used to visualize proliferating cells, apoptotic cells, neural pro-
genitors, and oligodendrocytes, respectively, together with the biotinylated
secondary antibodies from Vector ABC Elite kit (anti-mouse and anti-rabbit,
Vector), and developed using DAB Fast Kit (Sigma-Aldrich). Hematoxylin
(Tissue Tek) was used as a counterstain. COX-SDH immunohistological anal-
ysis was performed as previously described (Tyynismaa et al., 2005). Rabbit
anti-GFAP (1:400) and rabbit anti-calbindin (1:200) were used to visualize
astrocytes and subset of the periglomerular neurons of the olfactory bulb,
respectively, together with Alexa 594 chicken anti-rabbit secondary antibody.
Hoechst was used to stain nuclei. IgG from the primary antibody hosts was
used as a negative control for the staining. Analysis of optical density
(immunoperoxidase staining) or signal intensity (fluorescence staining) was
performed using ImageJ software.
Isolation of Hematopoietic Cells from Fetal Liver and Adult Bone
Embryos were harvested at E13–E15.5, and liver was dissected out and kept
on ice in 1% FBS. Fetal liver was mechanically suspended using scissors and
18–25 G needles, and the suspension was filtered through 40 mm filter (Dako)
and subjected to cell counting with crystal violet (Sigma-Aldrich). Adult mice
were sacrificed; their femoral and tibial bones were cut out and mechanically
cleaned. Bone marrow was flushed out with 1 ml of D-MEM supplemented
with penicillin-streptomycin and L-glutamine using insuline syringe, filtered
through 40 mm filter (Dako), and subjected to cell counting with crystal violet
(Sigma-Aldrich). The cells were immediately forwarded to analysis.
Flow Cytometry Analysis of Hematopoietic Tissues
The hematopoietic lineages derived from the fetal livers and adult bone
marrows were analyzed using FACSAria Cell Sorter (Beckton Dickinson) and
fluorescence-conjugated antibodies against Sca1, CD71, CD11b, CD34,
Ter119 (eBioscience), c-kit, and B220. All antibodies except Ter119 were
from BDPharMingen,and antibodies wereused 1mg/1 3106cells for staining.
CD16/CD32 blocker (0.25 mg/1 3 106cells) was used to inhibit possible non-
spesific binding of the antibodies, and isotype controls (1 mg/1 3 106cells)
were used to determine the background fluorescence of the labels. Thirty
thousand cells per cell line and antigen set were analyzed.
Hematopoietic Colony-Forming Assay
Presence of clonogenic hematopoietic progenitors in the fetal liver and
adult bone marrow was analyzed by culturing HPCs in MethoCult (StemCell
Technologies) for myeloerythroid progenitors according to the manufacturer’s
instructions. Duplicates were made from each sample.
Statistical significance between groups was determined by using Student’s
t test. Statistical analysis was performed when each group had at least three
samples. p < 0.05 was considered significant.
Supplemental Information includes two figures and Supplemental Experi-
mental Procedures and can be found with this article online at doi:10.1016/
We wish to thank the following funding agencies for support: Sigrid Juselius
Foundation, Jane and Aatos Erkko Foundation, the Academy of Finland,
University of Helsinki, Institute of Molecular Medicine Finland (FIMM), and
Biocentrum Helsinki (for A.S.); and Helsinki Biomedical Graduate School
(for K.J.A.). We thank Hanna Mikkola for advice and antibodies for HPC
FACS experiments and for very helpful discussions about hematopoietic
data. The authors are grateful for Anu Harju, Ilse Paetau, Tuula Manninen,
Markus Innila ¨, Minna Teerijoki, Ivana Bratic, Jarmo Palm, Susanna Lauttia,
is thanked for help in image analysis.
Received: June 7, 2011
Revised: October 24, 2011
Accepted: November 30, 2011
Published online: January 3, 2012
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