ARTICLE

doi:10.1038/nature11647
Towards germline gene therapy of
inherited mitochondrial diseases
Masahito Tachibana
1
, Paula Amato
2
, Michelle Sparman
1
, Joy Woodward
1
, Dario Melguizo Sanchis
1
, Hong Ma
1
,
Nuria Marti Gutierrez
1
, Rebecca Tippner-Hedges
1
, Eunju Kang
1
, Hyo-Sang Lee
1
, Cathy Ramsey
1
, Keith Masterson
2
,
David Battaglia
2
, David Lee
2
, Diana Wu
2
, Jeffrey Jensen
1,3
, Phillip Patton
2
, Sumita Gokhale
4
, Richard Stouffer
1,2
& Shoukhrat Mitalipov
1,2
Mutations in mitochondrial DNA (mtDNA) are associated with severe human diseases and are maternally inherited
through the eggs cytoplasm. Here we investigated the feasibility of mtDNA replacement in human oocytes by spindle
transfer (ST; also called spindlechromosomal complex transfer). Of 106 human oocytes donated for research, 65 were
subjected to reciprocal STand 33 served as controls. Fertilization rate in SToocytes (73%) was similar to controls (75%);
however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number
of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation
(38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid
karyotypes and contained exclusively donor mtDNA. The mtDNA can be efficiently replaced in human oocytes.
Although some ST oocytes displayed abnormal fertilization, remaining embryos were capable of developing to
blastocysts and producing embryonic stem cells similar to controls.
Mitochondrial DNA is localized in the cells cytoplasm, whereas
chromosomal genes are confined to the nucleus. Each cell may have
thousands of mtDNA copies, which may all be mutated (homo-
plasmy) or exist as a mixture (heteroplasmy). The clinical manifesta-
tions of mtDNAdiseases vary, but often affect organs and tissues with
the highest energy requirements, including the brain, heart, muscle,
pancreas and kidney
1
. The expression and severity of disease symptoms
depends on the specific mutation and heteroplasmy levels
1
.
An estimated prevalence of inherited mtDNA diseases is 1 in every
5,00010,000 live births, suggesting that, in the United States alone,
between 1,000 and 4,000 children are born every year with mtDNA
diseases
2,3
. Based on other estimates, the frequency of pathogenic
mtDNA mutations is even higher1 in 200 children inherit muta-
tions
4
. However, not all of these children develop the disease at birth,
because mtDNA mutations are present at low heteroplasmy levels.
At present, there are no cures for mitochondrial disorders and
available treatments only alleviate symptoms and delay disease pro-
gression. Therefore, several strategies for preventing transmission of
mtDNA mutations from mothers to their children have been actively
pursued.
One approach is to completely replace the mutated mtDNA of a
patients oocyte with the healthy mitochondrial genome from an
oocyte donated by another woman using spindle transfer (ST)
5
. The
technique isolates and transplants the chromosomes (nuclear genetic
material) from a patients unfertilized oocyte into the cytoplasm of
another enucleated egg, containing healthy mtDNA as well as other
organelles, RNA and proteins. A child born as a result of the ST pro-
cedure will be the genetic child of the patient but carry healthy mito-
chondrial genes from the donor. Our prior studies in a monkey model
demonstrated not only the feasibility of the ST procedure but also that
STis highly effective and compatible withnormal fertilization and birth
of healthy offspring
6
. This strategy has been considered clinically to be
a highly important future gene therapy to avoid transmission of serious
mitochondrial diseases (http://www.hfea.gov.uk/6372.html).
Here we present a comprehensive study demonstrating the feas-
ibility and outcomes of ST with human oocytes donated by healthy
volunteers. To measure success, we fertilized reconstructed oocytes
in vitro and assessed the normality of fertilization and embryo
development to blastocysts. In addition, we derived embryonic stem
cells (ESCs) and carried out detailed genetic analyses to assess efficacy
of gene replacement and possible chromosomal abnormalities asso-
ciated with ST. We also conducted additional studies in a rhesus
macaque model to investigate the feasibility of using cryopreserved
oocytes for ST and postnatal development of ST offspring.
Mitochondrial DNA replacement in human oocytes
Seven volunteers (aged 2132 years) underwent ovarian stimulation
and a total of 106 mature metaphase II (MII) oocytes were retrieved
(range of 728, or a mean of 15 oocytes per donor cycle). Participants
were synchronized in three separate experiments, so that at least two
fresh oocyte cohorts were available on the same day for reciprocal ST.
We selecteda total of 65 MII oocytes for the STprocedure and33 served
as non-manipulated controls (Fig. 1a). We successfully transferred the
spindle surrounded by a membrane and a small amount of cytoplasm
(karyoplast) between64 oocytes (98%; Fig. 1a, b). Sixty oocytes survived
fertilization by intracytoplasmic sperm injection (ICSI; 94%) and 44
formed visible pronuclei (73%). These outcomes were similar to the
results for controls; 32 oocytes survived ICSI (97%) and 24 (75%)
formed pronuclei (Fig. 1b). Microscopic evaluations determined that
almost half of STzygotes (21/44, 48%) contained normal two pronuclei
and two polar bodies (2PN/2PB) (Fig. 2a). However, the remaining ST
zygotes had an irregular number of pronuclei and/or polar bodies
(Fig. 2a). Abnormal fertilization was also observed in the intact control
group, albeit at a lower incidence (3/24, 13%).
1
Division of Reproductive & Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, 505 NW185th Avenue, Beaverton, Oregon 97006, USA.
2
Division of
Reproductive Endocrinology, Department of Obstetrics andGynecology, OregonHealth &Science University, 3181SWSamJacksonPark Road, Portland, Oregon97239, USA.
3
Center for Womens Health,
Oregon Health &Science University, 3181 SWSamJackson Park Road, Portland, Oregon 97239, USA.
4
University Pathologists, LLC, Boston University School of Medicine; Roger Williams Medical Center,
Providence, Rhode Island 02908, USA.
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Blastocyst formation rate in the normally fertilized STgroup (13/21,
62%) was statistically similar to controls (16/21, 76%). However, the
majority of abnormal STzygotes arrested, with only 26%(6/23) reach-
ing blastocysts (Supplementary Table 1). Interestingly, blastocyst
development of 3PN/1PB zygotes was noticeably higher (4/11, 36%)
than that of other abnormally fertilized ST groups (Supplementary
Table 1).
Derivation and genetic analysis of human ESCs
To provide additional insights into the developmental competence of
ST-produced human embryos and to obtain sufficient material for
molecular and cytogenetic analyses, we derived ESCs from blasto-
cysts. Nine ESC lines (HESO lines) were established from 16 control
blastocysts (56%; Fig. 1b). This ESC derivation rate is significantly
higher than currently reported for embryos donated by IVF patients
7
.
Similarly, 13 ST blastocysts developed from normally fertilized
zygotes produced five ESC lines (38%; HESO-ST lines). We also pla-
ted four ST blastocysts that originated from 3PN/1PB zygotes and
derived one ESC line (25%; Supplementary Table 1).
The ESCs derived from the ST embryos had normal morphology
and were indistinguishable from controls (Supplementary Fig. 1a, b).
All ESCs expressed standard pluripotency markers, including OCT-4,
SOX2, SSEA-4, TRA-1-81 and TRA-1-60 (Supplementary Fig. 1a, b).
Following injection into immunodeficient mice, experimental ESCs
formed teratoma tumours consisting of cells and tissues representing
all three germ layers (Supplementary Fig. 1c). Detailed analysis of
nuclear DNA using microsatellite markers confirmed that all
HESO-ST lines inherited their chromosomes from the spindle donor
oocytes (Fig. 3a, Table 1 and Supplementary Fig. 2a, d). Analysis of
mtDNA confirmed that all HESO-ST cell lines derived their mtDNA
from the cytoplast donors (Fig. 3b, Table 1 and Supplementary Figs
2b, e and 3)
6,8
.
As reported previously
6
, small amounts of mtDNA are usually co-
transferred with the karyoplast during the ST, causing a lowcarryover
heteroplasmy. In clinical situations this may result in the transmission
of mutant mtDNA to ST embryos and children. Therefore we con-
ducted both qualitative and quantitative mtDNA assays to determine
the degree of mtDNA carryover in ST embryos and ESC lines. We
identified mtDNA sequence differences between oocyte donors and
unique restriction enzyme recognition sites for restriction-fragment
length polymorphism (RFLP) assay
6
. For example, mtDNA of the
egg donor 1 (spindle contributor for HESO-ST2, -ST3 and -ST4)
possessed a unique EcoRVdigestion sequence GATATC(Supplemen-
tary Fig. 4a). In contrast, egg donor 2 (mtDNA contributor) carried a
single nucleotide polymorphismGATACCprecluding enzyme recog-
nition. The results confirmed that mtDNA in ST cell lines was exclu-
sively derived from the cytoplast donors with no detectable mtDNA
carryover (Table 1 and Supplementary Figs 2c and 4a). We also used
more sensitive ARMSqPCR (amplification refractory mutation
systemquantitative polymerase chain reaction) that enables the mea-
surement of heteroplasmy below 1%
9,10
. The mean mtDNA carryover
in ST oocytes and embryos was 0.5%(s.d. 60.4; range 00.9%) and in
HESO-ST cell lines was 0.6% (s.d. 60.9; range 01.7%) (Supplemen-
tary Fig. 4b and Supplementary Table 2). These results are consistent
with our previous data from a nonhuman primate
6
and suggest neg-
ligible mtDNA carryover in ST offspring.
Cytogenetic analyses, using G-banding, indicated that all five HESO-
ST lines derived from normal ST embryos contained diploid male or
female karyotypes, with no evidence of detectable numerical or struc-
tural chromosomal abnormalities (Table 1 and Supplementary Fig. 5).
However, two out of nine lines derived from control embryos had
numerical aberrations. Notably, HESO-6 carried a 47 XYY karyo-
type, whereas HESO-9 was 45 XO (Supplementary Table 3 and
Egg donor A
Egg donor B
ST ICSI Pronuclear
formation
Blastocyst ESC isolation
MII spindle
Karyoplast
Cytoplast
*
*
n = 33 32
24
16
9
97%
75%
67%
56%
Controls
n = 64
60
44
19
6
94%
73%
43%
32%
a
b
0
0
100%
80
60
40
20
100%
80
60
40
20
Fertilization Blastocycts ESCs Survived
after ICSI
No. of
oocytes
Spindle transfer
Figure 1 | Experimental design and main outcomes after ST with human
oocytes. a, Oocytes were retrieved from two unrelated donors and spindle
chromosomal complexes were reciprocally exchanged. Reconstructed oocytes
were fertilized by ICSI and monitored for in vitro development to blastocysts
and ESCs. Images in boxes depict a human mature MII oocyte with the spindle
visualized under polarized microscope (left), isolated cytoplast and karyoplast
(middle) and intact spindles inside recipient cytoplasts after transfers (right,
arrowheads). Original magnifications: left and middle, 3200; right, 3100.
Asterisks indicate first polar bodies. b, Experimental outcomes after ST in
human oocytes. The top and bottom graphs represent fertilization, blastocyst
and ESC isolation rates for intact control and ST embryos, respectively. No
statistical differences were found betweenSTandcontrols insurvival after ICSI,
fertilization, blastocyst development and ESC derivation rates (P.0.05).
2PN/2PB
48%
3PN/1PB
25%
6.8%
3PN/2PB
1PN
6.8%
Multiple PN (>3)
6.8%
Abnormal two-cell
6.8%
Metaphase II
Anaphase II
a
b
Figure 2 | Abnormal pronuclear formation and spindle morphology in
human ST zygotes. a, Proportion of normally fertilized zygotes with two
pronuclei and two polar bodies (2PN/2PB) compared with abnormal zygotes
(3PN/1PB, 3PN/2PB, 1PN, multiple PN and two-cell) after ST. b, Integrity of
meiotic spindles inhumanSToocytes depicting normal metaphase II (top), and
premature progression to the anaphase II (bottom).
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Supplementary Fig. 6). G-banding revealed that HESO-ST6, the cell
line derived fromabnormally fertilized STzygote, contained abnormal
triploid female chromosome complement (Supplementary Fig. 5). In
addition, detailed microsatellite analysis of nuclear DNA in this cell
line confirmed the presence of three alleles for most short tandem
repeat (STR) loci (Supplementary Table 4). On the basis of allele inher-
itance, we concluded that the triploid karyotype was caused by reten-
tion of the genetic material of the second polar body. This was
consistent with observation of the extra pronucleus but lack of the
second polar body in the zygote.
Abnormal fertilization in human zygotes produced by ST
Abnormal fertilization observed in some human ST oocytes was
unexpected because this was not observed in monkey studies
6
.
Therefore, additional experiments were conducted to investigate pos-
sible underlying mechanisms. Genetic analysis of the HESO-ST6 cell
line hinted that some ST oocytes and embryos retain extra chromo-
somes that are normally extruded into the second polar body. This
was likely to be caused by sub-optimal conditions during the ST
procedure that disturbed spindle integrity. Initially, we focused on
the effect of cytochalasin B (CB), a microfilament inhibitor known
to inhibit cytokinesis and to block the second PB extrusion in
oocytes
11,12
. CB is used acutely during the ST procedure and oocytes
are thoroughly rinsed before fertilization, but residual CB could inter-
rupt the second PBextrusion. Therefore, we extended incubation time
between ST and ICSI, or decreased CB concentration. However,
abnormal fertilization persisted even in the absence of CB (Sup-
plementary Tables 5 and 6), indicating that abnormal meiotic segrega-
tion is not likely to be caused by CB exposure.
We next addressed whether oocyte polarity during displacement of
spindles leads to abnormal meiosis. Typically, spindles in MII oocytes
are adjacent to the first polar bodies. However, during the ST pro-
cedure, karyoplasts are reintroduced on the opposite side (referred to
as 180 degree)
5,6
. We reintroduced spindles next to or at the 90 degree
from the first PB (Supplementary Fig. 7). However, ST zygotes had
similar pronuclear abnormalities (Supplementary Fig. 7).
Lastly, we reasoned that human meiotic spindles may undergo
premature activation during the ST manipulations leading to incom-
plete resumption of meiosis after fertilization. Spindle morphology
and meiotic stage was analysed in intact and ST human oocytes
following immunolabelling with a- and b-tubulin. Analysis demon-
strated that in some ST oocytes spindles already progressed to the late
anaphase II, whereas all control oocytes maintained uniform meta-
phase II (Fig. 2b).
Oocyte cryopreservation before ST
Current ST protocols use fresh oocytes and require that both patient
and healthy mtDNA egg donors undergo synchronous retrievals.
However, it is difficult to manage the same-day egg retrievals owing
to differences in the ovarian cycle and responses to gonadotropins. In
addition, an equal number of patient and donor eggs retrieved would
be ideal to avoid oocyte wastage. Therefore, oocyte freezing, storage
and thawing will be critical for clinical applications of the ST. Recent
advances inoocyte vitrification procedures suggest that cryopreserved
human MII oocytes can be used in clinical IVF practice with the same
efficiency as fresh eggs
13,14
. To evaluate the feasibility of using cryo-
preserved oocytes for ST, we turned to the nonhuman primate model.
We tested a commercially available vitrification kit (CRYOTOP) and
determined that survival and recovery of rhesus macaque MII oocytes
post-thaw is high. After ISCI, 72% formed pronuclei, but only 6%
developed to blastocysts (P ,0.05) (Table 2). Thus, this cryopreser-
vation method compromises blastocyst development, because blasto-
cyst formation of fresh oocytes from the same cohort was 52%
(Table 2)
6,9
.
We next conducted reciprocal STbetween fresh and frozen-thawed
monkey oocytes and examined fertilization and embryo development
(Supplementary Fig. 8). When fresh spindles were transplanted
into vitrified cytoplasts, fertilization after ICSI was impaired (50%)
Table 1 | Genetic analysis of human ESCs derived from ST blastocysts
Cell line HESO-ST-2 HESO-ST-3 HESO-ST-4 HESO-ST-5 HESO-ST-6 HESO-ST-7
Nuclear donor Donor 1 Donor 1 Donor 1 Donor 4 Donor 3 Donor 6
Cytoplast donor Donor 2 Donor 2 Donor 2 Donor 3 Donor 4 Donor 7
Fertilization 2PN/2PB 2PN/2PB 2PN/2PB 2PN/2PB 3PN/1PB 2PN/2PB
Karyotype (passage no.) 46 XX, P4 46 XX, P7 46 XY, P7 46 XX, P4 69 XXX, P4 46 XY, P3
Nuclear DNA origin (by STR) Donor 1 Donor 1 Donor 1 Donor 4 Donor 3 Donor 6
mtDNA origin Donor 2 Donor 2 Donor 2 Donor 3 Donor 4 Donor 7
mtDNA carryover (RFLP) Undetectable Undetectable Undetectable Undetectable Undetectable NT
mtDNA carryover (ARMSqPCR) 0.20% 0.01% 1.70% NT NT NT
NT, not tested.
Egg donor 1
Egg donor 2
HESO-ST2
HESO-ST3
HESO-ST4
Sperm donor
Egg donor 1
Egg donor 2
HESO-ST2
HESO-ST3
HESO-ST4
XY
XX
XX
XX
XX
XY
191/193
191/199
187/201
193/199
191/193
193/199
227/229
AME D7S513 D6S1691
211/223
225/225
223/229
211/227
211/229
16282 C/T 16329 C/T
a Nuclear DNA genotyping
b MtDNA genotyping
Figure 3 | Genetic analysis of ESCs derived from human ST embryos.
a, Nuclear DNA origin of HESO-ST2, -ST3 and -ST4 determined by
microsatellite parentage analysis. The microsatellite markers for D7S513 and
D6S1691 loci demonstrate that the nuclear DNA in these ESC lines was from
the egg donor 1 (the spindle donor). b, mtDNA genotyping by direct
sequencing show that the mtDNA in HESO-ST2, -ST3 and -ST4 is originated
from the egg donor 2.
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compared to controls (91%) (Table 2). Moreover, all embryos in this
ST group arrested before reaching blastocysts, whereas 57% controls
progressed to blastocysts. These ST results were similar to those seen
with frozen-thawed intact controls (Table 2). However, when spindles
from vitrified oocytes were transferred into fresh cytoplasts, fertiliza-
tion (88%) and blastocyst formation (68%) rates were similar to fresh
controls (Table 2). These results indicate that vitrification causes
damage primarily within the cytoplasm rather than to the spindle
apparatus.
Tofurther evaluate developmental potential, we platedsixSTblasto-
cysts derived from vitrified spindles onto feeder cells and established
two ESClines (33%). We transplanted four STblastocysts fromvitrified
spindles into a recipient that resulted in the timely birth of a healthy
infant (Supplementary Fig. 8).
Postnatal development of monkey SToffspring
Although the technical feasibility of mtDNA replacement is docu-
mented for human embryos and ESCs, questions remain regarding
whether a mismatch between mtDNA and nuclear DNA haplotypes
may cause mitochondrial dysfunctions in ST children
15
. To address
these concerns, we conducted a 3-year follow-up study on monkey ST
offspring born in 2009 (ref. 6).
The growth and development of four healthy infants following ST
procedure was evaluated during the postnatal period (Supplementary
Fig. 9a). Their overall health, including routine blood and body-
weight measurements monitored from birth to 3 years were com-
parable to age-matched controls. The values for haemoglobin, red
blood cell and white blood cell counts, mean corpuscular volume
and haemoglobin concentrations were all within normal ranges
(Supplementary Table 7). We also measured blood chemistry and
arterial blood gas parameters and demonstrated that metabolic status
of ST offspring is comparable to controls (Supplementary Table 7).
In addition, the body-weight gain for the ST juvenile monkeys was
similar to that of age-matched controls (Supplementary Fig. 9b). We
also confirmed that ATP levels and mitochondrial membrane poten-
tial (DYm) in skin fibroblasts were similar to those of controls
(Supplementary Fig. 10). Finally, there were no significant changes
in mtDNA carryover and heteroplasmy in blood and skin samples
with age (Supplementary Fig. 9c).
Discussion
This report summarizes our effort to test an mtDNA replacement in
unfertilized human oocytes, initially developed and optimized in a
monkey model. The results demonstrate that the ST procedure can be
performed with high efficiency in human oocytes. Manipulated
oocytes also supported high fertilization rates similar to those of con-
trols. However, approximately half of the human ST zygotes had
abnormal fertilization, primarily as a result of excessive pronuclear
numbers. This was an unexpected outcome that was not observed
with monkey oocytes. Our follow-up studies indicated that this is
caused by the failure to complete meiosis and segregate chromosomes
into the second PB, probably owing to premature activation. A set of
haploid genetic material is normally discarded during asymmetrical
cell division into the second PB, while the other half forms the female
pronucleus. By genetic analysis of ESCs derived from abnormally
fertilized zygote, we confirmed the triploid nature and presence of
two sets of female chromosomes.
The spindle-chromosomal apparatus in MII oocytes is an extremely
sensitive structure that can easily be perturbed by physical or chemical
manipulations. Our initial attempts to isolate and transplant monkey
MII spindles were unsuccessful owing to similar problems with spon-
taneous resumption of meiosis
6
. Procedures were optimized to avoid
this negative outcome and current ST protocols allow maintenance of
an intact MII spindle and normal fertilization. It seems that human
MII oocytes are more sensitive to spindle manipulations and further
improvements and optimizations will be required for future clini-
cal applications. Maintenance of meiotic spindles in MII oocytes
is dependent on the activity of M-phase-specific kinases including
maturation-promoting factor (MPF) and mitogen-activated protein
kinase
16
. Under normal conditions, sperm entry triggers degradation
of kinase activities and chromosome segregation mediated by oscilla-
tions of intracellular Ca
21
concentrations
17
. However, an influx of
calciuminduced by mechanical or chemical manipulations can induce
parthenogenetic activation of oocytes and resumption of meiosis
18
.
Thus, ST manipulations in a medium without Ca
21
or supplementa-
tions with MG132 could potentially avoid problems with spontaneous
activation
19,20
.
Morphological evaluation of fertilization and early detection of
abnormal pronuclear and/or polar body formation seems to be critical
to separate normal and abnormal ST embryos. Blastocyst develop-
ment and ESC isolation in normally fertilized ST zygotes were similar
to controls. We also confirmed that all ESC lines derived from these
ST embryos are karyotypically normal.
Two of the nine ESC lines (22%) derived from non-manipulated
oocytes also showed chromosomal abnormalities. Because aberra-
tions were confined to the sex chromosomes (47 XYY and 45 XO),
it is possible that this was induced by sperm carrying either two Y
chromosomes or no Y chromosome.
Despite the risk of abnormal pronuclear formation and aneuploidy
in a portion of ST zygotes, embryo development and ESC isolation
rates in normal STzygotes are comparable to intact controls. Based on
our estimates of retrieving on average 12 MII oocytes, 35% normal
(2PN/2PB) fertilization rates, and 60% blastocyst development, at
least two ST blastocysts suitable for transfers can be generated during
a single cycle for each patient.
The safety of the ST procedure is also dependent on the amount of
mutated mtDNA co-transferred with spindles. Importantly, mtDNA
carryover in ST embryos and ESC lines is technically undetectable or
below 1%. In most patients with mtDNA diseases, a threshold of 60%
or higher of mutated mtDNA must be reached for clinical features to
appear. Thus, it is unlikely that low mtDNA carryover during ST
would cause disease in children. Segregation of mutated mtDNA to
specific tissues during development and ageing may hypothetically
result in a significant accumulation of the mutant load. However,
analysis of mtDNA carryover in monkey ST offspring discovered
Table 2 | Fertilization and embryo development of frozen rhesus oocytes
Experiment Group n Survived after ST (%) Survived after ICSI (%) Fertilized (%) Blastocysts (%)
1 Fresh oocytes 32 NA 30 (94) 29 (97) 15 (52)*
Vitrified oocytes 26 NA 25 (96) 18 (72) 1 (6)
2 Control fresh oocytes 34 NA 33 (97) 30 (91){ 17 (57){
Fresh cytoplasts
Vitrified spindles
36 34 (94) 32 (94) 28 (88){ 19 (68){
Vitrified cytoplasts
Fresh spindles
35 35 (100) 34 (97) 17 (50) 0
* Blastocyst rate statistically different from that for vitrified oocytes (P,0.05).
{ Fertilized rate statistically different from that for vitrified cytoplasts with fresh spindles (P,0.05).
{ Blastocyst rate statistically different from that for vitrified cytoplasts with fresh spindles (P,0.05).
Data were analysed using analysis of variance (ANOVA).
NA, not applicable.
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no detectable mtDNA segregation into different tissues
9
. In addition,
there were no changes in heteroplasmy levels during postnatal
development of monkeys. Thus, carryover, segregation and tissue-
specific accumulation of mutant mtDNA molecules in ST children
seem unlikely to be major concerns.
Birth of a healthy monkey infant after oocyte freezing marks an
important milestone in applying the ST technology to patients.
Transplantation of vitrified spindles into fresh cytoplasms yields the
best results, comparable to controls. However, fertilization of vitrified
cytoplasts evenwithfreshspindles was compromised. These remarkable
findings indicate that the damage after cryopreservation is confined
mainly to the eggs cytoplasm, not to the chromosomes and spindles
as commonly believed
21
. Our observations also reveal another un-
expected potential clinical application of the ST technique, suggesting
that spindles insub-optimally cryopreserved oocytes canbe rescued by
transplanting into fresh cytoplasts.
Follow-up postnatal studies in four monkeys produced by ST pro-
vide convincing evidence that oocyte manipulation and mtDNA
replacement procedures are compatible with normal development.
These monkeys were derived by combining nuclear and mtDNAfrom
the two genetically distant subpopulations of rhesus macaques.
Mitochondrial and nuclear genetic differences between these mon-
keys are considered to be as distant as those between some different
primate species
22
, thus imitating haplotype differences between
humans. Concerns have been raised that nuclear and mtDNA incom-
patibilities between mtDNA patients and cytoplast donors may cause
a mismatch and mitochondrial dysfunctions in ST children even in
the absence of mutations
15
. On the basis of our long-term observa-
tions, it is reasonable to speculate that nuclearmtDNA interactions
are conserved within species.
Pioneering work in nonhuman primates is critical for the develop-
ment, and safety and efficacy evaluations, of new treatments
23,24
. It is
important that scientists and clinicians further optimize ST protocols
for human oocytes and ensure that these procedures are safe. It is also
crucial that the US Food and Drug Administration initiates careful
reviewof these newdevelopments. Such oversight will be important to
establish safety and efficacy requirements and guide clinical trials.
Current US National Institutes of Health funding restrictions sur-
rounding these innovative reproductive technologies will also require
amendments to support federally funded clinical trials.
METHODS SUMMARY
The study protocols were approved by both the Oregon Health & Science
University Embryonic Stem Cell Research Oversight Committee and the
Institutional Review Board.
Mature oocytes were donated by volunteers and ST procedures were carried
out as described
5,6
. Oocytes were fertilized, cultured to blastocysts and used for
ESC isolation. Detailed methods are described in Supplementary Information at
www.nature.com/nature.
Received 9 June; accepted 3 October 2012.
Published online 24 October 2012.
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Supplementary Information is available in the online version of the paper.
Acknowledgements The authors would like to acknowledge the Oregon Health &
Science University (OHSU) Embryonic Stem Cell Research Oversight Committee and
the Institutional Review Board for providing oversight and guidance. We thank oocyte
and spermdonors and staff at the Womens Health Research Unit at the Center for
Womens Health, University Fertility Consultants and Reproductive Endocrinology &
Infertility Division at the Department of Obstetrics & Gynecology of Oregon Health &
Science University for their support and procurement of human gametes. The Division
of Animal Resources, Surgery Team, Assisted Reproductive Technology & Embryonic
Stem Cell Core, Endocrine Technology Core, Imaging & Morphology Core, Flow
Cytometry Core and Molecular &Cellular Biology Core at the Oregon National Primate
Research Center provided expertise and services for the nonhuman primate research.
HamiltonThorneInc., donatedanXYClonelaser systemfor thisstudy. Wearegrateful to
W. Sanger and D. Zaleski for karyotyping services, C. Penedo for microsatellite analysis
and J. Henneboldfor consulting onmetabolic assays. We are also indebtedto A. Steele,
R. Cervera Juanes and E. Wolff for their technical support. The human oocyte/embryo
research was supported by grants fromthe OHSUCenter for Womens Health Circle of
Giving and other OHSU institutional funds, as well as the Leducq Foundation. The
nonhuman primate study was supported by grants from the National Institutes of
Health HD063276, HD057121, HD059946, EY021214 and 8P51OD011092.
Author Contributions M.T., P.A., J.J. and S.M. conceived the study, designed
experiments and wrote institutional review board protocols. P.A., M.S. and N.M.G.
coordinated recruitment of participants. P.A., K.M., D.B., D.L., D.W. and P.P. performed
ovarianstimulationandoocyte recovery. M.T. conducted ST micromanipulations. M.S.,
K.M. andS.M. performedICSI. M.T., M.S., J.W., D.M.S., N.M.G., R.T.-H. andE.K. conducted
ESC derivation and characterization. S.G. analysed teratoma tumours. M.T., H.M. and
D.M.S. performed DNA/RNA isolations, metabolic and mtDNA analyses. C.R., M.T.,
M.S.,H.-S.L., R.S. and S.M. conducted monkey studies. M.T., R.S., J.J., P.P. and S.M.
analysed data and wrote the paper.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Readers are welcome to comment on the online version of the paper. Correspondence
and requests for materials should be addressed to S.M. ([email protected]).
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