Corrected 19 October 2021. See full text.
RES EARCH
◥ spermatogenesis upon transplantation into
REVIEW SUMMARY
GAMETOGENESIS
Mammalian in vitro gametogenesis
Mitinori Saitou* and Katsuhiko Hayashi*
BACKGROUND: Mammalian germ cells differ-
entiate into oocytes in females and spermato-
zoa in males. An oocyte and a spermatozoon
fuse to form a zygote and then develop to
create a new individual, thereby transmitting
their genetic and epigenetic information to
the next generation. During development,
germ cells undergo epigenetic reprogram-
ming and programming to acquire totipo-
tency upon fertilization, maintain genome
information with high fidelity, and, paradoxi-
cally, create genome diversity through meiotic
recombination.
Intensive efforts have been made to explore
the molecular and systems-level mechanisms
underlying these distinctive functions that germ
cells acquire during their development. On the
basis of the knowledge obtained through such
endeavors, notable progress has been made
over the past decade on research aiming at
reconstituting germ cell development in vitro
using pluripotent stem cells (PSCs), including
embryonic stem cells (ESCs) derived from
preimplantation embryos and induced PSCs
iPSCs
Germline
Soma
Die away
in one
generation
Genetic/epigenetic integrity?
Ethical issues?
In vitro gametogenesis. IVG aims to recreate germ cell development in vitro using PSCs, including iPSCs
from somatic cells. Human IVG realization requires further efforts and will create new opportunities in
reproductive biology research and medicine. ART, assisted reproductive technologies.
Saitou et al., Science 374, 47 (2021) 1 October 2021
(iPSCs) generated from somatic cells through
transcription factor (TF)–induced reprogram-
ming. This process, referred to as in vitro game-
togenesis (IVG), is not only providing a basis for
further exploration of the mechanisms of germ
cell development but also creating prospects for
innovative medical applications.
ADVANCES: Mouse PSCs (mPSCs) have been
induced into mouse primordial germ cell–
like cells (mPGCLCs), the founding germ cell
population. Oogenesis has been reconstituted
by culturing mPGCLCs with mouse embry-
onic ovarian somatic cells [reconstituted ovaries
(rOvaries)], and the resultant oocytes have
produced offspring. This system has revealed
key mechanisms for oocyte development, in-
cluding the TFs engendering oocyte-like growth
in mouse ESCs. The mPGC-to-spermatogonia
development has been reconstituted by cul-
turing mPGCLCs with mouse embryonic tes-
ticular somatic cells [reconstituted testes
(rTestes)], and the resultant spermatogonia
have propagated in vitro and contributed to
PGCLCs
In vitro Spermato- Immature
gonia oocytes
gametogenesis
Oocytes
Sperm
Basic mechanisms
Modeling infertility
Improving ART
testes. Furthermore, the oogenic fate determi-
nation pathway has been reconstituted with-
out the use of ovarian somatic cells, revealing
the mechanisms for epigenetic reprogramming,
oogenic fate determination, and meiotic entry.
Human PSCs (hPSCs) have been induced
into human PGCLCs (hPGCLCs), and the mech-
anism for hPGC(LC) specification has been
clarified and shown to involve key TFs, their
hierarchy of actions, and their regulatory
wiring, all of which are divergent from those
for mPGC(LC) specification. hPGCLCs have
been shown to undergo epigenetic reprogram-
ming and differentiate into early oocytes or
prospermatogonia by culturing with mouse
embryonic ovarian or testicular somatic cells
(xenogeneic rOvaries or rTestes), respectively,
establishing a foundation for human IVG.
More recently, mPSCs have been induced into
mouse embryonic ovarian somatic cell–like
cells fully capable of supporting mPGCLC-
Downloaded from https://www.science.org on December 05, 2023
based oogenesis, paving a way for generating
corresponding cells in humans and other spe-
cies, including endangered species, and hence
for robustly promoting their IVG.
OUTLOOK: The proof-of-concept mouse IVG
and the basic framework of human IVG
have been established, creating opportuni-
ties for exploring the mechanisms of mouse
and human germ cell development, includ-
ing those that have been both technically
and ethically difficult to address. IVG tech-
nology can be applied to other animals, in-
cluding endangered species, enabling species
preservation. Realizing human IVG will re-
quire further advances but will create possi-
bilities for diagnosing and modeling infertility,
exploring its remedies, and improving ar-
tificial reproductive technologies, thereby
advancing reproductive medicine. Human
IVG could also be extended for reproduc-
tive application, but before that would be
deemed permissible, analyses must include
appropriate “normality” assessments of the
IVG-derived animal models—ideally primate
models—and genetic and epigenetic assess-
ments of hPSCs and the resultant gametes.
Once all technological concerns have been
resolved, it will be crucial to hold society-wide
discussions about whether to use IVG-derived
gametes for human reproduction, because
such an application would change our un-
derstanding of human origins and the conti-
nuity of life.
▪
The list of author affiliations is available in the full article online.
*Corresponding author. Email: saitou@anat2.med.kyoto-u.ac.jp
(M.S.); hayashik@hgs.med.kyushu-u.ac.jp (K.H.)
Cite this article as M. Saitou, K. Hayashi, Science 374,
eaaz6830 (2021). DOI: 10.1126/science.aaz6830
READ THE FULL ARTICLE AT
https://doi.org/10.1126/science.aaz6830
1 of 1
 Corrected 19 October 2021. See full text.
RES EARCH

◥ spermiogenesis to form haploid spermatozoa

REVIEW (10). Notably, in mice, but not in humans, a
primary SSC line, referred to as germline stem
GAMETOGENESIS cells (GSCs), with a robust capacity for self-

Mammalian in vitro gametogenesis

renewal in vitro and for spermatogenesis upon
transplantation into testes, has been derived,
prompting investigations into the properties
Mitinori Saitou1,2,3* and Katsuhiko Hayashi4,5* of SSCs and GSCs (11). Testicular somatic
cells, particularly Sertoli cells, play pivotal
Germ cells differentiate into sexually dimorphic gametes, oocytes, and spermatozoa, which unite to form roles in supporting male germ cell develop-
new individuals. Accordingly, germ cell development entails intricate regulations of genome functions ment (4, 8, 10).
for genetic and epigenetic inheritance. The past decade has seen considerable advances in in vitro The sexually dimorphic composition of the
gametogenesis (IVG), which aims to recreate germ cell development from pluripotent stem cells (PSCs) sex chromosomes has key effects on germ cells
in culture. Mouse PSCs can be induced into functional oocytes and spermatozoa, whereas human PSCs in a cell-intrinsic manner, although it primar-
can be induced into early oocytes and prospermatogonia, promoting mechanistic understanding of ily affects the sex of the somatic cells. The XX
mammalian germ cell development. The prospect for inducing human gametes with appropriate composition is beneficial for the growth and
functions has been heightened, and such advances will create possibilities in reproductive medicine, development of oocytes (12), whereas XY is
including modeling infertility to explore remedies. The use of IVG-derived gametes for human critical for the development of spermatogonia
reproduction will require careful legal and ethical discussions. as well as for the spermatogenesis (13). The
general principles of germ cell development

M
are well conserved among mammals. How-
ammalian germ cells develop via intri- ation to demethylate DNA genome-wide and ever, there are critical species differences in

Downloaded from https://www.science.org on December 05, 2023

cate and precise processes to generate
sexually dimorphic gametes, oocytes
and spermatozoa. Gametes fuse and
develop to form new individuals that
inherit genetic and epigenetic information
from the parents. Anomalies in germ line de-
velopment result in infertility and genetic or
epigenetic disorders in the offspring. The past
decade has seen great progress in research to
generate germ cells in vitro from pluripotent
stem cells (PSCs), including embryonic stem
cells (ESCs) and induced pluripotent stem cells
(iPSCs) that are derived from somatic cells.
Here, we will discuss the current state of mam-
malian in vitro gametogenesis (IVG) research
(1), key challenges driving the research forward,
and critical considerations associated with the
applications of IVG research.
Germ cell development in mammals
Germ cells arise as primordial germ cells (PGCs)
from pluripotent cells during early mamma-
lian development (1, 2) (Fig. 1). PGCs migrate
through the developing hindgut endoderm
and mesentery and colonize the embryonic
gonads, where they proliferate as oogonia in
females or gonocytes in males and initiate sex-
ually dimorphic development. In contrast to
somatic cells, PGCs undergo epigenetic repro-
gramming during their migration and prolifer-
1
Institute for the Advanced Study of Human Biology (WPI-ASHBi),
Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501,
Japan. 2Department of Anatomy and Cell Biology, Graduate
School of Medicine, Kyoto University, Yoshida-Konoe-cho,
Sakyo-ku, Kyoto 606-8501, Japan. 3Center for iPS Cell
Research and Application (CiRA), Kyoto University, 53
Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
4 (SSCs). These cells essentially halt their develop-
Department of Developmental Stem Cell Biology, Faculty of
Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku,
Fukuoka 812-8582, Japan. 5Department of Germline Genetics,
Graduate School of Medicine, Osaka University, 2-2 Yamada-
oka, Suita, Osaka 565-0871, Japan.
*Corresponding author. Email: saitou@anat2.med.kyoto-u.ac.jp
(M.S.); hayashik@hgs.med.kyushu-u.ac.jp (K.H.)
alter histone modification, leading to the era-
sure of parental imprints and X chromosome
reactivation in females (2, 3).
In the ovaries of XX embryos, oogonia re-
ceive signals from nascent granulosa cells (so-
matic cells that surround developing oogonia)
or the ovarian environment to differentiate
into primary oocytes and enter into meiotic
prophase (4–7) (Fig. 1). The oocytes proceed
into the diplotene stage of meiotic prophase
and form a tight complex with granulosa cells,
creating primordial follicles. At this stage, most
of the primordial follicles pause their devel-
opment. They resume follicle growth, that is,
oocyte growth coupled with expansion of the
granulosa cell layers, around puberty in re-
sponse to hormonal cues and develop into
fully matured antral follicles. Mature oocytes
possess the maternal genome and epigenome
(6, 7). Upon ovulation and fertilization with
spermatozoa, the antral follicles complete
their first and second meiotic divisions, respec-
tively, to form a zygote. Ovarian somatic cells,
particularly granulosa cells, exert an essential
function in promoting female germ cell de-
velopment (4, 6, 7).
In the testes of XY embryos, nascent Sertoli
cells (somatic cells that surround developing
gonocytes and form seminiferous tubules) or
the testicular environment signal gonocytes
to differentiate into prospermatogonia, which
undergo mitotic arrest and acquire the paternal
epigenome, including imprints and a defense
against transposon activities (3–5, 8, 9) (Fig. 1).
After birth, prospermatogonia differentiate into
spermatogonia or spermatogonial stem cells
ment until puberty, at which time they initiate
robust waves of spermatogenesis, the process
by which a mitotic stem cell gives rise to hap-
loid gametes, consisting of mitotic amplifi-
cations followed by meiotic divisions and
the time scale required for key developmen-
tal transitions, developmental synchronicity,
and the signaling, transcriptional, and meta-
bolic properties of developmentally homolo-
gous cells (1, 2, 5) (Fig. 1).
IVG in mice
Mouse PGC-like cells (mPGCLCs)
During development, mouse PGCs (mPGCs)
originate from the most proximal epiblast at
around embryonic day 6.5 (E6.5) as Blimp1
(also known as Prdm1)–positive cells in re-
sponse to bone morphogenetic protein 4
(BMP4) from the extraembryonic ectoderm
(14–17) (Figs. 1 and 2). Proto-oncogene protein
WNT3, which is activated in the epiblast by
BMP signaling, is also essential for mPGC
specification (17) (Fig. 2). The three transcription
factors (TFs) BLIMP1, PRDM14, and TFAP2C
are essential for mPGC specification, which
involves repression of a somatic program, re-
acquisition of the pluripotency network, and
epigenetic reprogramming (16, 18, 19) (Fig. 3).
The epiblast cells from around E5.25 to E6.25
show competence to differentiate into mPGCs
in response to BMP4 (17).
There are at least two distinct states of
pluripotency in culture: naïve pluripotency
and primed pluripotency (20) (Fig. 2). Mouse
ESCs (mESCs) derived from the inner cell mass
of the blastocysts bear naïve pluripotency and
contribute to chimeras and germ cells, and
they are typically cultured with inhibitors for
fibroblast growth factor and mitogen-activated
protein kinase (FGF/MEK) and glycogen syn-
thase kinase 3b (GSK3b) in the presence of
leukemia inhibitory factor (LIF) (a culture
condition known as 2i+LIF) (21–23). mESCs
show properties similar to those of the peri-
implantation epiblast (~E4.5) (21, 24) (Fig. 2).
In contrast, mouse epiblast stem cells (mEpiSCs)
derived from the postimplantation epiblast

Saitou et al., Science 374, eaaz6830 (2021) 1 October 2021 1 of 9

 Corrected 19 October 2021. See full text.
RES EARCH | R E V I E W

Primary oocytes Antral Oocytes (MII)

(Prophase I)
Pm Pr
Sc

ExE Al
ICM Mesoderm
Ovary Granulosa

SG SC
R-ST El-ST
PGCs SG (TypeB)
(TypeA)
TE Pro-SG Sertoli
Epiblast VE Migrating PGCs PGCs in
genital ridges PGCs in gonads Testis Spermatozoa
Mouse
Pro-SG

Fertilization 2cell Blastocyst E5.0 E6.5 E8.5 E10.5 E12.5 E13.5 Birth Puberty
1week
Fertilization 2cell Blastocyst 2wks 3wks 5wks 8wks 10wks 20wks Birth Puberty
Oogonia/Primary oocytes
(Prophase I) Antral Oocytes (MII)
Amnion
Human PGCs in Pm Pr Sc
PGCs
york sac
ICM Epiblast Al

Ovary Granulosa
SG SC R-ST El-ST
York sac
Pro-SG SG (TypeB)
Hypoblast (TypeA)
TE ExMC Migrating PGCs Sertoli
PGCs in gonads Testis Spermatozoa
Gonocytes/Pro-SG
(Mitotic arrest)

Downloaded from https://www.science.org on December 05, 2023

Oogonia/Gonocytes/Pro-SG Sertoli cells Interstitial cells (Male) Myoid cells
Primary oocytes Granulosa cells Interstitial cells (Female) Epithelial cells

Fig. 1. Scheme for germ cell development in mice and humans. Schematic
representations of germ cell development in mice (top) and humans (bottom).
Embryonic days (E) in mice and developmental weeks (wks) in humans are
shown, with 1-week periods marked with symbols, as indicated. In ovaries,
granulosa cells surround oogonia and primary oocytes, creating primordial
follicles (a single squamous granulosa cell layer). They undergo follicle growth,
that is, oocyte growth coupled with expansion of the granulosa cell layers,
and develop into primary (a single cuboidal granulosa cell layer), secondary (several
granulosa cell layers), and antral follicles (multiple granulosa cell layers with antral
cavity). In testes, Sertoli cells surround gonocytes and prospermatogonia and
form seminiferous tubules, in which male germ cell development progresses both
during development and in adults. ICM, inner cell mass; TE, trophectoderm; ExE,
extraembryonic ectoderm; Al, allantois; PGCs, primordial germ cells; Pm, primordial
follicles; Pr, primary follicles; Sc, secondary follicles; MII, meiosis II; Pro-SG,
prospermatogonia; SG, spermatogonia; SC, spermatocyte; R-ST, round spermatids;
El-ST, elongating spermatids.

at ~E5.5 to E6.5 show primed pluripotency
with a biased differentiation potential and con-
tribute little, if any, to chimeras under conven-
tional conditions (25, 26). mEpiSCs self-renew
in the presence of activin and FGF and exhibit
properties similar to the epiblast after E6.5
(27) (Fig. 2).
mEpiSCs are refractory to differentiation
into germ cells (28). A strategy to induce mESCs
into competent epiblast-like cells (mEpiLCs)
was explored, leading to the finding that an
~2-day stimulation by activin and FGF induces
mESCs into mEpiLCs bearing properties sim-
ilar to the epiblast at E5.5 to E6.0 (28) (Figs. 2
and 4). In response to BMP4, mEpiLCs robustly
differentiated into mPGC-like cells (mPGCLCs)
with properties similar to mPGCs at the migrat-
ing stage (~E9.5). mPGCLC induction faithfully
recapitulated the mPGC specification process
in vivo. Male mPGCLCs contributed to sper-
matogenesis when transplanted into testes of
neonatal mice lacking endogenous spermato-
genesis (28); female mPGCLCs contributed to
oogenesis when aggregated with mouse em-
bryonic ovarian somatic cells (reconstituted
ovaries, or rOvaries) and transplanted under
the ovarian bursa of immune-deficient mice
(29); the resultant spermatozoa and fully
grown oocytes produced fertile offspring
(28, 29) (Fig. 4). These processes were rep-
licated using both male and female mouse
iPSCs (miPSCs) (28, 29) (Fig. 4), establishing
a foundation for IVG.
The state exhibited by mEpiLCs or pregas-
trulating mouse epiblast has been proposed
to represent a formative state, an immediately
precursory state for multilineage differenti-
ation, including germ cell differentiation, in
response to external cues (30) (Fig. 3). Con-
sistently, mEpiLCs have a distinctive epigenome
with abundant bivalent histone modifications
in key developmental genes, which are poised
for timely expression (31, 32). Recently, by
mainly modulating activin and WNT signal-
ing, a formative state has been stably captured
in vitro (33, 34).
The mPGCLC induction system has been
useful in analyzing the mechanisms of mPGC
specification (35–39) (Fig. 5). For example, the
finding that T (also known as TBXT), a down-
stream effector of WNT signaling, activates
Blimp1 and Prdm14 illuminates a mechanism
that bridges the external signaling and in-
ternal transcription network for mPGC(LC)
specification (35) (Fig. 3). Homeobox protein
OTX2, a TF essential for anterior neuroecto-
derm specification, has been shown to act as
a repressor of mPGC(LC) specification, whose
absence leads to an extended competence of
the epiblast and mEpiLCs to adopt the germ
cell fate (38). Moreover, it has been shown that
miPSCs generated from infertile mice with sex
chromosome trisomy, such as XXY (Klinefelter
syndrome), preferentially lose the extra sex
chromosome, and the resultant euploid XY
miPSCs differentiate into mPGCLCs and
contribute to spermatogenesis and offspring,
providing a strategy for overcoming infer-
tility with abnormal sex chromosome con-
stitutions (40).
In vitro oogenesis
On the basis of organ and reaggregation cul-
ture of embryonic ovaries (41–43), a method-
ology for rOvary culture has been established
(44) (Fig. 4). rOvaries are cultured under an
air–liquid interface condition for in vitro de-
velopment (IVD; ~3 weeks), followed by in vitro
growth (~2 weeks). During IVD, mPGCLCs dif-
ferentiate into oogonia and then into oocytes
with an entry into the prophase of meiosis I
(MI) and form oocytes at the secondary follicle
stage. Under the in vitro growth conditions,
they differentiate into fully grown oocytes at
the antral follicle stage, which can be induced
into oocytes at the meiosis II (MII) stage through
in vitro maturation (Fig. 4). The MII oocytes
from both mESCs and miPSCs can be fertilized
to form offspring (44), establishing a proof-of-
concept for IVG.
Compared with in vivo oogenesis, however,
the fidelity of the in vitro oogenesis procedure
is compromised. The developmental rate of

Saitou et al., Science 374, eaaz6830 (2021) 1 October 2021 2 of 9

 Corrected 19 October 2021. See full text.
RES EARCH | R E V I E W

Naive Formative PGC specification Primed

pressure yielded dormant oocytes, demon-
strating a critical involvement of biophysical
Rodent Implantation BMP4 mechanisms for primordial follicle recruit-
ment (51, 53).
Mouse A P
Embryo The in vitro oogenesis system also revealed
development
WNT3 that XO and XY oocytes in rOvaries with XX
Rat gonadal somatic cells exhibit impaired devel-
(mESC/iPSC) bFGF+activin A bFGF opment owing to frequent chromosomal mis-
2i+LIF/3i
(rESC/iPSC) (mEpiLC (transient state)) (mEpiSC)
Ying et al.
Buehr et al.
Hayashi et al. Tesar et al. pairing during the prophase of MI, followed
Brons et al.
PSC state Li et al. AloXR (activin A+XAV939+BMS493) by oocyte apoptosis (54). The impaired de-
(mFSCs)
Kinoshita et al. velopment was more profound in XY oocytes,
Conv. (mESC/iPSC) FTW (bFGF+activin A+CHIR) partially owing to an inhibitory effect on
(mXPSCs)
Yu et al. oogenesis of a Y-linked translational initiator,
Eif2s3y, that promotes spermatogenesis (54, 55).
Primate This system offers a strategy for circumvent-
Implantation WNT3A
BMP4 ing the infertility caused by sex chromosome
Embryo
development A P disorders, for example, screening of chemicals
that nullify a meiotic checkpoint by sex chro-
Human 5i/L/A (hESC/iPSC) 4i (hESC/iPSC) mosome heteroploidy in oocytes.
Theunissen et al. Gafni et al.
Cynomolgus AloXR (activin A+XAV939+BMS493)
Moreover, a set of TFs sufficient for oocyte
t2i/L/Gö (hESC/iPSC)
monkey Takashima et al. (hFSCs) growth has been identified: simultaneous ex-
Kinoshita et al.
pression of NOBOX, FIGLA, LHX8, TBPL2,

Downloaded from https://www.science.org on December 05, 2023

PSC state FTW (bFGF+activin A+CHIR)
(hXPSCs) SOHLH1, STAT3, SUB1, and DYNLL1, each of
Yu et al.
which is essential for oocyte growth, induced
Conv. (hESC/iPSC) female mESCs into oocyte-like cells when ag-
AITS+IF20 (cyESC) gregated with ovarian somatic cells, and no-
Sakai et al.
tably, such cells were capable of fertilization,
Others demonstrating that oocyte growth per se can
(Rauber’s layer species) Implantation
BMP2/4 be independent from preceding germ cell de-
Rabbit Embryo velopment (56) (Fig. 5), which extends a pre-
development A P
WNT3A vious finding that oocyte growth is dissociable
from meiosis (57). This work paves the way for
Pig Epiblast Hypoblast/Primitive endoderm Amnion Nascent Mesoderm
Trophectoderm Anterior Visceral Endoderm Primordial Germ Cells Extra-embryonic Mesenchyme the TF-based generation of developmentally
competent ooplasm.
Fig. 2. Divergence of early development and spectrum of pluripotency in vivo and in vitro in various
mammals. Schematic illustrations of the development of a portion of preimplantation and early In vitro spermatogenesis
postimplantation embryos in rodents (mice and rats, top), primates (humans and monkeys, middle), and A methodology for the reaggregation culture
other representative mammals (rabbits and pigs, bottom). PSCs cultured under various conditions in vitro are of male mPGCLCs with embryonic testicular
aligned with embryonic cells bearing the closest similarity in their transcriptomes. See text and specified somatic cells [reconstituted testes (rTestes)]
references for details. Conv., conventional ESC culture media with serum and LIF; bFGF, basic fibroblast has been explored (58) (Fig. 4). The rTestes
growth factor; AloXR, low activin A, XAV939, and RAR inhibitor; mFSC, mouse formative stem cells; FTW, FGF, cultured under an air–liquid interface condi-
TGFb, and WNT; mXPSC, mouse chimera and PGC dual-competent PSCs; AITS+IF20, AlbuMax, insulin, tion self-organized seminiferous tubule-like
transferrin, selenoprotein, IWR1, bFGF 20 ng/ml; A, anterior; P, posterior. structures, in which mPGCLCs differentiated
into gonocytes, prospermatogonia, and then
in vitro–derived two-cell embryos to term (~3%; and in vitro oogenesis will be an important spermatogonia (~3 weeks). Compared with
mESC-derived: 1.2 to 3.9%; miPSC-derived: challenge. in vivo development, in vitro development was
0.3 to 0.9%) is ~1/20 to 1/100 that of in vivo Notwithstanding these limitations, the protracted and less efficient, and mPGCLC-
embryos (44). In vitro oogenesis is charac- in vitro oogenesis system has been instrumen- derived spermatogonia failed to undergo mei-
terized by a higher incidence of homologous tal in clarifying key mechanisms for oocyte osis, although some progressed to the prophase
chromosome mispairing, lower mitochondrial development. In the ovary, most oocytes re- of MI after a prolonged culture (~8 weeks).
DNA amounts, and precocious resumption main dormant as primordial follicles, ensuring The potential of mPGCLC-derived spermato-
of the first meiotic division. These defects oocyte storage and reproductive longevity (6, 7). gonia was demonstrated by the derivation of
could stem from both an inadequate quality In rOvaries, however, few dormant oocytes GSC-like cells (GSCLCs) in culture (58) (Fig. 4).
of mPSCs and suboptimal in vitro culture (primordial follicles) are formed, and almost GSCLCs were similar to GSCs in morphology,
conditions. Regarding the former, mPSCs— all oocytes develop alongside somatic cells to expansion capacity, and transcriptome and
particularly female mPSCs—cultured under form secondary follicles (29, 44). Transcriptome contributed to spermatogenesis in adult testes,
the 2i+LIF condition show epigenetic abnor- analyses revealed that oocytes in primordial with the resultant spermatozoa giving rise to
malities, including genome-wide DNA demeth- follicles in vivo are enriched with genes for fertile offspring. The spermatogenesis efficiency
ylation (45, 46), and a prolonged culture leads response to hypoxia and extracellular matrix, of GSCLCs, however, was substantially lower
to their compromised developmental poten- which is consistent with the fact that they than that of GSCs (3 out of 15 GSCLC lines
tial (47, 48). iPSCs bear additional epigenetic are located around the ovarian cortex bearing contributed to spermatogenesis, and the three
abnormalities derived from somatic cells and an avascular and collagen-rich histology contributing lines showed ≦~20% spermato-
reprogramming errors (49, 50). Thus, improve- (51, 52). Accordingly, rOvary culture under a genesis colonies, compared with ~100% sper-
ments of culture conditions for female mPSCs hypoxic condition or with extra-hydrostatic matogenesis by GSCs) owing to an aberrant

Saitou et al., Science 374, eaaz6830 (2021) 1 October 2021 3 of 9


RES EARCH | R E V I E W
Mouse
BMP
WNT
T EOMES
Modulate
Pluripotency
Blimp1
Germ cell
development
Prdm14
Epigenetic
reprogramming
Tfap2c
Somatic
differentiation
epigenome programming. Nonetheless, GSCLC-
derived offspring appear grossly normal, sug-
gesting that appropriate epigenotypes are
selected during spermatogenesis or early de-
velopment or that animals indeed bear epi-
mutations whose physiological manifestations
escape detection (58). This study demonstrates
an in vitro induction of SSC activity from mPSCs
and points to the importance of epigenetic re-
programming and programming for generat-
ing an appropriate paternal epigenome, whose
more faithful reconstitution warrants further
investigation.
There are several reports claiming to have
achieved the generation of haploid spermatid-
like cells from mPSCs in vitro [see (59)], but
these studies did not recapitulate the in vivo
processes and did not perform an appropri-
ate evaluation of the key intermediate events,
including epigenetic reprogramming and
programming. The in vitro reconstitution of
the meiotic divisions for spermatogenesis re-
mains a critical challenge.
mPGCLC differentiation under
defined conditions
Attempts have been made to promote mPGCLC
differentiation under defined conditions free
from gonadal somatic cells. It has been shown
that mPGCLCs are propagated to ~50-fold
in 1 week in the presence of forskolin and
rolipram, which stimulate cyclic adenosine
monophosphate signaling, on m220 feeders
that provide a membrane-bound form of stem
cell factor (60). During expansion, while main-
Saitou et al., Science 374, eaaz6830 (2021)
Corrected 19 October 2021. See full text.
Fig. 3. Signaling and
transcriptional network
for PGC specification
Human in mice and humans.
Schematic representations
GATA3 of the signaling and
GATA2
transcriptional network
for PGC specification
in mice (left) and humans
(right). Arrows indicate
regulatory relationships
among signaling and
transcription factors.
SOX17
BLIMP1
TFAP2C
taining their transcriptome, mPGCLCs exhibit
genome-wide DNA demethylation in a manner
consistent with a replication-coupled, passive
demethylation, reaching a level equivalent to
germ cells at E13.5 (~5%), which bear the lowest
DNA methylation levels throughout the germ-
line cycle and initiate either an oogenic or a
spermatogenic program (46, 60). This work
demonstrates that genome-wide DNA demeth-
ylation and germ cell sex determination are
genetically dissociable.
Accordingly, it has been demonstrated that
BMP signaling is a primary mechanism that
induces mPGC(LC)s into the oogenic pathway,
including meiotic entry (61, 62). In mPGC(LC)s
that have undergone epigenetic reprogram-
ming, BMP (BMP2 and BMP5 are expressed in
granulosa cells) and its downstream effector,
ZGLP1, a conserved TF with GATA-like zinc
fingers, activate key oogenic programs, such
as those for chromatin modification, meiotic
cell cycle, and folliculogenesis. On the other
hand, retinoic acid (RA), which has been re-
garded as a key for female germ cell specifi-
cation [see (4) for review], assists in the overall
oogenic program maturation as well as the
PGC program repression. mPGCLCs induced
by BMP or ZGLP1 progress at least up to the
pachytene stage of meiotic prophase (61, 62).
Consistently, recent reports have shown that
in mice lacking all RA receptor isoforms or all
RA synthesizing genes, female germ cells still
enter into meiosis (63, 64).
A concept has been introduced that germ
cells require “licensing” for their sex differen-
1 October 2021
tiation (65). A replication-coupled genome-wide
DNA demethylation, which erases methyla-
tions on key genes for oogenesis and spermato-
genesis, and BMP signaling that induces the
oogenic programs fulfill the mechanisms for
the oogenic fate licensing. The TF-induced
oocyte-like growth in female mESCs (56), which
bear a globally demethylated epigenome simi-
lar to that of E13.5 germ cells (45, 46), is consist-
ent with this idea. Uncovering the mechanism
for implementing the spermatogenic fate re-
mains a key challenge.
IVG in humans
Origin of the human germline: Lessons from
cynomolgus monkeys
A translation of the strategies used in mouse
IVG into humans could be one of the reason-
able means for realizing human IVG. For this
purpose, two key issues require careful consid-
eration. hPSCs are more similar to mEpiSCs
than to mESCs in morphology, signaling, and
Downloaded from https://www.science.org on December 05, 2023
transcriptional and epigenetic profiles (20, 66).
Also the origin of human germ cells has been
intractable, as it occurs early in postimplanta-
tion, a stage ethically and technologically diffi-
cult to analyze (Figs. 1 and 2).
Studies using cynomolgus monkeys, a pri-
mate closely related to humans, have provided
insights into these two issues (67, 68). Single-
cell transcriptome analyses revealed that epi-
blast cells in cynomolgus monkeys change their
transcriptome considerably upon implantation
and thereafter, while generating gastrulat-
ing cells (~E13), maintain a relatively stable
transcriptome, and cynomolgus monkey ESCs
(cyESCs) bear a transcriptome highly similar
to that of postimplantation late (E16 or E17)
and early (E12 or E13) epiblast cells (67) (Fig.
2). Regarding the expression of a gene set
(~500 genes) that characterizes the develop-
mental transitions of epiblast cells from the
naïve state to the primed state, hPSCs are high-
ly similar to cyESCs and hence to the post-
implantation late or early epiblast (67) (Fig. 2).
A recent report on single-cell transcriptomes of
a human gastrulating embryo (E16 to E19) also
showed that hPSCs represent a postimplanta-
tion epiblast state (69). At the same time, ef-
forts have been made to realize the derivation of
naïve hPSCs. Two culture systems called 5i/L/A
(chemical inhibitors for MEK, GSK3b, RAF,
SRC/LCK and ROCK, plus LIF and activin) and
t2i/L/Gö (chemical inhibitors for MEK, GSK3b
and PKC, plus LIF) convert primed hPSCs
into those bearing transcriptional and epi-
genetic features of preimplantation epiblast
(70, 71) (Fig. 2). These cultures, however, are
not optimal, because they show relatively
weak transcriptional concordance with the
preimplantation epiblast (67, 69) and exhibit
epigenetic and karyotypic instability (70, 71).
Nonetheless, these cells, but not primed hPSCs,
could be induced into the trophectoderm
4 of 9
 Corrected 19 October 2021. See full text.
RES EARCH | R E V I E W

Derivation of mouse Derivation of

mouse oocytess Derivation of
PGCLCs (female) mouse spermatozoa
Hikabe et al.
Hayashi et al. Derivation of ESCs/iPSCs Derivation of Ishikura et al.
ESCs/iPSCs mouse spermatid
-like cells mouse germline ESCs
stem cell-like cells
Derivation of mouse Zhou et al.
PGCLCs (male) Ishikura et al.
Hayashi et al. ESCs EpiLCs ESCs
EpiLCs
EpiLCs
ESCs/iPSCs

GSCLCs
EpiLCs PGCLCs EpiLCs
PGCLCs
EpiLCs PGCLCs

Reaggregation with
Reaggregation with E12.5 ovarian soma
E12.5 ovarian soma PGCLCs PGCLCs Expansion in vitro

PGCLCs Injection

Co-culture with newborn Reaggregation with

testicular soma E12.5 testicular soma
Transplantation Transplantation
into testes into ovaries Reaggregation with
E12.5 testicular soma
Spermatozoa

Spermatozoa Round spermatid Oocytes GSCLCs

Oocytes

Mouse

2011 2012 2015 2016 2018 2020 2021

human Rhesus monkey Cynomolgus human iPSCs
ESCs/iPSCs human iPSCs human iPSCs ESCs monkey ESCs

Downloaded from https://www.science.org on December 05, 2023

iMeLCs
iMeLCs iMeLCs PGCLCs
Pre-induction iMeLCs PGCLCs
Human /4i culture PGCLCs PGCLCs
Sakai et al.
PGCLCs PGCLCs Transplantation Derivation of Reaggregation with
Cynomolgus/ Reaggregation into testes cynomolgus E12.5 mouse
Rhesus monkey with E12.5 mouse
monkey PGCLCs
testicular soma
ovarian soma
Irie et al. Sasaki et al.

Derivation of human PGCLCs

Early oocytes
Pro-spermatogonia
-like cells
Immature spermatogonia
Hwang et al.
Sosa et al.
Derivation of rhesus Derivation of human
Yamashiro et al. monkey PGCLCs pro-spermatogonia
Derivation of human
oogonia/primary oocytes

Fig. 4. Progress of IVG research in mice and humans. A research timeline of the progress of key IVG research in mice (top) and humans and monkeys (bottom).
Top right inset image shows elongated spermatids expressing the Ddx4-RFP transgene (red) and peanut agglutinin (green) counterstained with DAPI (white). Bottom
inset image shows early oocytes expressing DDX4 (magenta) and SCP3 (cyan).

EpiLCs PGCLCs
Blimp1, Prdm14, Tfap2c Transplantation into testes
Nakaki et al.
Spermatozoa
EpiLCs PGCLCs
Nanog
Murakami et al. Primary oocytes
PGCLCs
Zglp1 Oocyte-like cells
(w/o meiosis)
ESCs Nagaoka et al.
Nobox, Figla, Lhx8, Tbpl2
Skip Sohlh1, Stat3, Sub1, Dynll1
Hamazaki et al.
Mouse Epiblast

ICM Epiblast/Amnion
PGCs Primary oocytes Primordial
(Prophase I) follicles MII oocytes
ESCs (4i) PGCLCs
SOX17, BLIMP1
Kobayashi et al.
Human
Oogonia
iMeLCs PGCLCs
GATA3 or GATA2
SOX17, TFAP2C Reaggregation
Kojima et al. with E12.5 mouse
ovarian soma

Fig. 5. TF-based IVG research in mice and humans. Schematic illustrations of the progress of key TF-based IVG research in mice (top) and humans (bottom). The
cell types in which indicated TFs are expressed and the resultant cell types are shown, with an alignment with in vivo cell types bearing the closest similarity.

Saitou et al., Science 374, eaaz6830 (2021) 1 October 2021 5 of 9


RES EARCH | R E V I E W
lineage, indicating that they capture the po-
tential of preimplantation embryonic cells
(72, 73). Accordingly, recent studies showed
that under appropriate three-dimensional cul-
ture conditions, naïve hPSCs self-organize into
human blastocyst-like structures with epiblast-,
trophectoderm-, and hypoblast-like cell differ-
entiation (74, 75).
A histological study showed that cynomolgus
monkey PGCs (cyPGCs) that express key mark-
ers [SOX17 and TFAP2C (see below)] originate
in the amnion, which differentiates from the
epiblast after implantation (68) (Fig. 2). cyPGCs
are observed in the amnion from around E11 to
E17, initially dorsally and later posteriorly, and
increase their numbers and migrate in the
hindgut. The amnion itself expresses not only
BMP4 and its effectors, but also mesodermal
markers, including T, fulfilling the properties
of cells destined toward the germ cell fate (68).
Experiments involving an extended culture of
cynomolgus monkey or human preimplanta-
tion embryos have shown that cyPGCs or hu-
man PGCs (hPGCs) indeed originate from an
amnion-like structure (76, 77). Although the
possibility that cyPGCs and hPGCs originate
from the epiblast has not been excluded, these
findings represent an evolutionary divergence
of developmental pathways among mammals.
It seems reasonable to expect that primates,
including humans, adopt a strategy for germ
cell specification that reflects their structur-
al characteristics, which are different from
those in mice and other mammals. The hPGC
transcriptome obtained at the earliest stage
(E16 to E19) may shed new light on their origin
and properties (69).
hPGCLC induction and mechanism of
hPGC(LC) specification
Methodologies to induce human PGCLCs
(hPGCLCs) from hPSCs have been explored.
hPSCs cultured under a 4i condition [chemical
inhibitors for MEK, GSK3b, p38, and c-Jun
N-terminal kinase (JNK)] (78), which represent
a perigastrulating epiblast-like state (67, 79)
(Fig. 2), are induced into hPGCLCs in response
to BMP signaling (79) (Fig. 4). Alternatively,
hPSCs cultured under a conventional condi-
tion are first induced into incipient mesoderm-
like cells (iMeLCs) and then into hPGCLCs by
BMP signaling (80) (Fig. 4). hPGCLCs in both
studies show gene expression properties sim-
ilar to early hPGCs and to each other (79, 80).
Subsequent studies have shown that hPGCLCs
have the potential to undergo epigenetic repro-
gramming, a hallmark of germ cells, and differ-
entiate into early oocytes or prospermatogonia
(81, 82) (Fig. 4), indicating that hPGCLCs are
a bona fide in vitro counterpart of hPGCs (see
below).
These studies show that the primed plu-
ripotency of hPSCs is distinct from that of
mEpiSCs, because the latter show little com-
Saitou et al., Science 374, eaaz6830 (2021) 1 October 2021
Corrected 19 October 2021. See full text.
petence for germline differentiation (28). hPSCs
appear to bear an extended primed pluripo-
tency competent to generate all the cell types
that emerge from the epiblast after implan-
tation, including the amnion and germ cells.
Accordingly, recent studies have reported a
potential reconstitution of amnion differen-
tiation from hPSCs (83).
With the hPGCLC induction system, the
mechanism of hPGC(LC) specification has been
explored (79, 80, 84–88). It has been shown
that SOX17, which is known as a key TF for
endoderm differentiation but is dispensable
for PGC specification in mice, acts as one of
the most upstream TFs for hPGC(LC) specifi-
cation, and BLIMP1 functions downstream of
SOX17 (79, 84) (Fig. 3). SOX17 appears to be
critical in PGCs not only among primates
(68, 89, 90) but also in evolutionarily distant
mammals such as pigs (91), suggesting that the
mouse may have evolved a distinct strategy for
PGC specification. Indeed, hPGCLC specifica-
tion involves key TFs, their hierarchy of ac-
tions, and their regulatory network distinct
from mPGC(LC) specification (84, 86–88)
(Fig. 3), and the whole transcriptomes for
hPGCLC specification diverge from those for
mPGC(LC) specification (80, 90, 92).
Moreover, whereas mPGC(LC) specification
is directly coupled with epigenetic reprogram-
ming (31, 46, 60, 93), hPGCLC specification is
genetically dissociable from epigenetic reprog-
ramming (94). Upon mPGC(LC) specification,
BLIMP1, PRDM14, and TFAP2C repress key
machineries for de novo and maintenance DNA
methylation (Dnmt3a, Dnmt3b, and Uhrf1) and
create a state with little DNA methylation ac-
tivity, leading to a replication-coupled, pas-
sive genome-wide DNA demethylation upon
mPGC(LC) proliferation (31, 46, 60, 93, 95, 96).
In contrast, although hPGCLC specification
accompanies a down-regulation of DNMT3A/
3B, it maintains UHRF1, which recruits DNMT1
into replication foci, at a substantial level, and
hPGCLCs retain their genome-wide DNA meth-
ylation profiles and levels upon their prolif-
eration (94), highlighting another layer of TF
network divergence between mouse and human
PGC(LC) specification.
Human early oocyte and
prospermatogonia induction
Advances in omics technologies have created
rapid progress in our understanding of the
transcriptome and epigenetic dynamics as-
sociated with human germ cell development
(5, 82, 97–104). Human germ cells use a dis-
tinctive gene regulatory network and, over a
period of a few months, undergo epigenetic
reprogramming with a cell-to-cell variation.
This includes genome-wide DNA demethyla-
tion, with imprint erasure and X chromosome
reactivation in females and with evolution-
arily young transposons relatively resistant to
demethylation, which might result in trans-
generational epigenetic inheritance.
Human female fetal germ cells (FGCs) can
be classified into four cell types [mitotic (hPGCs
and oogonia), RA-responsive, meiotic, and
oogenesis], whereas male FGCs can be classified
into at least three [migrating (hPGCs), mitotic
(gonocytes), and mitotic arrest (fetal pros-
permatogonia)] (5, 100). RA-responsive FGCs
emerge starting at around week 11 and express
genes such as ZGLP1, STRA8, and ANHX at
high levels while repressing early hPGC genes,
yet they show low-level expression of meiotic
genes including SYCP1, SPO11, and PRDM9;
meiotic FGCs that express such meiotic genes at
high levels emerge starting at around week 14,
suggesting that it should take ~3 weeks for
RA-responsive FGCs to mature into meiotic
FGCs. In mice, the corresponding transition
occurs much more quickly (in under ~2 days),
and the RA-responsive state has not been rec-
ognized as a distinct entity. Consistent with
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the histological observations (105, 106), even
at week 26, all four female FGC types coexist in
individual female embryos, whereas two male
FGC types coexist in single male embryos
(100), demonstrating substantial develop-
mental asynchrony of human FGCs.
An aggregate culture of hPGCLCs with mouse
embryonic ovarian somatic cells [xenogeneic
rOvary (xrOvary)] has been used to induce
hPGCLCs into human germ cells at later de-
velopmental stages (81) (Fig. 4). In xrOvaries,
hPGCLCs interact with mouse granulosa cells
and induce maturation of their characteristics,
albeit at slower speed and with lower efficiency
than hPGCs in vivo: At around 70 days in cul-
ture, hPGCLC-derived cells differentiate into
oogonia or gonocyte-like cells, expressing key
genes including DAZL, DDX4, and PIWIL2,
and notably, at around 120 days, they acquire
a state highly similar to RA-responsive FGCs
(Fig. 4). Accordingly, they undergo genome-
wide DNA demethylation, imprint erasure, and
partial X reactivation (81). These findings es-
tablish a foundation for human IVG. Similarly,
it has been shown that an aggregation culture
of hPGCLCs with mouse embryonic testicular
somatic cells [xenogeneic rTestis (xrTestis)]
differentiates hPGCLCs into gonocytes and
then into prospermatogonia in a time frame
similar to that of the differentiation of hPGCLCs
to oogonia to RA-responsive FGCs (82).
The mechanism underlying hPGCLC matu-
ration into early oocytes and prospermatogonia
in xrOvaries and xrTestes, respectively, is un-
known. The defined culture system for hPGCLC
expansion should serve as a platform to explore
such mechanisms, including a mechanism to
induce epigenetic reprogramming (94). The
impact of the X chromosome states in female
hPSCs on the hPGCLC specification and matura-
tion requires investigation. hPSCs typically bear
one active and one inactive X chromosome
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RES EARCH | R E V I E W
(Xa and Xi, respectively). However, the Xi
in hPSCs is unstable, with the expression of
XIST (X-inactive specific transcript), a long
noncoding RNA important for X inactivation,
being progressively repressed upon hPSC pas-
sages, leading to a partial derepression of Xi
(an X-eroded state, Xe) (107–109). There is a
trend in which female hPSCs show lower effi-
ciency for hPGCLC induction than do male
hPSCs (92, 110), and the degree of the X chro-
mosome reactivation in oogonia might re-
flect the original X chromosome state in hPSCs
and influence the maturation of oogonia into
oocytes (81).
Key challenges
IVG has established itself as a key research
area in biomedical science, one facing a num-
ber of salient challenges in coming years.
Although in vitro oogenesis in mice has been
achieved, PSC-based in vitro spermatogenesis
remains a key challenge. There have been re-
ports describing an organ culture of immature
testicular fragments in mice, by which sperma-
togonia and SSCs go through proper meiotic
divisions and develop into spermatids with full
developmental potential (111). With this system,
GSCs can be used as an appropriate donor (112).
The most recent version of this system uses a
multilayered microfluidic chamber, where sem-
iniferous tubules spread into a sheetlike struc-
ture, allowing an equal distribution of nutrients
and oxygen across the tissue masses (113). Ac-
cordingly, a potential strategy for an mPSC-
based in vitro spermatogenesis could be to
combine an improved rTestis-based GSCLC
induction with an organ culture of GSCLC-
transplanted testes. Indeed, using such strategy,
a recent study has demonstrated the in vitro
reconstitution of whole male germ cell devel-
opment from mouse PSCs (Fig. 4) (114).
To further extend IVG in humans, use of a
xenogeneic system would not be ideal, because
species differences would preclude efficient
human gametogenesis in such systems. Greater
success would be expected if human fetal or
neonatal gonadal somatic cells were used to
form human rOvaries or rTestes. The isola-
tion and use of adequate amounts of human
fetal gonadal somatic cells at an appropriate
developmental stage, however, represents a
technical and ethical challenge. Similarly, using
gonadal material from the same species would
also improve efficiency for IVG in endangered
animals (115). A strategy to fundamentally re-
solve this limitation is to induce such cells from
PSCs. Indeed, in line with the advancements
in our understanding of the mechanism of
gonadal development (116, 117), fetal ovarian
somatic cell–like cells fully capable of supporting
oogenesis have been induced from mPSCs (118).
This study will serve as a basis to supply cells
equivalent to embryonic gonadal somatic cells
in humans and other species in coming years.
Saitou et al., Science 374, eaaz6830 (2021) 1 October 2021
Corrected 19 October 2021. See full text.
Concomitantly, the development of organ
(ovary or testis) culture systems that allow the
maturation of immature germ cells in humans
and other species represents a critical chal-
lenge. Meanwhile, transplantation-based strat-
egies could be a first option to demonstrate
the feasibility of generating PGCLC-derived
gametes in primates and other species, par-
ticularly in males (89, 90, 119–121). Efforts to
expand SSCs in vitro (GSCs) in humans and
other species are also critical.
An ultimate goal of IVG research will be to
reconstitute the entirety of gametogenesis
using defined factors only. The processes of
mPGCLC specification, propagation, epigenetic
reprogramming, and female sex determination
have been recapitulated (60–62), and hPGCLCs
have been propagated to ~106-fold under de-
fined conditions (94). Moreover, key TFs driv-
ing the corresponding respective processes as
well as oocyte growth have been determined
(36, 56, 62, 88). With further understanding
of the mechanisms for germ cell development,
including those for later processes such as sper-
miogenesis in males and full oocyte growth in
females, the extent to which the gametogenesis
processes are recapitulated under defined con-
ditions would expand further.
As an alternative source for haploid cells,
haploid ESCs have been generated from andro-
genetic or parthenogenetic (bearing only the
paternal or maternal genome, respectively)
haploid eggs in mice and humans, providing
a platform to explore the function of genes in
relevant developmental pathways [see (122, 123)
for review]. On the other hand, mouse offspring
derived from such cells often die prematurely
in part because of aberrant imprints, indicating
genetic or epigenetic vulnerability of haploid
mESCs (122, 123). Further studies are required
for appropriate applications of haploid mouse
and human ESCs.
Critical considerations
The realization of human IVG will create pos-
sibilities in reproductive medicine, such as
opportunities for diagnosing and modeling
infertility and genetic and epigenetic disor-
ders, exploring their remedies, and improving
culture methodologies for assisted reproduc-
tive technologies (124). If deemed legally and
ethically permissible, the use of IVG-derived
human gametes for producing offspring would
require careful assessments and intensive studies.
It would be essential that the “normality” of the
IVG-derived animal models be strictly assessed,
although thus far such mouse models appear
grossly normal (28, 29, 44, 58–60, 93). It should
be noted that many of the mouse embryos gen-
erated from oocytes via in vitro oogenesis died
prenatally owing to abnormal development,
and even those born apparently normally bore
heavier placentae (44), suggesting that the
surviving animals may have cryptic anom-
alies. Thus, IVG-derived animals should be
scrutinized with respect to many parameters,
including gene expression, epigenetic proper-
ties, behavior, longevity, and disease suscep-
tibility. Given that some epigenetic mutations
are heritable (125), these evaluations should be
performed for several generations. It is also
essential to create IVG-derived animal models
more relevant to human physiology, such as
IVG-derived macaques, and to perform similar
normality assessments.
Concomitantly, the genetic and epigenetic
quality of hPSCs and the resultant gametes
require careful assessment. It has become in-
creasingly evident that somatic cells accumu-
late de novo mutations widely (126, 127) and
that iPSCs acquire additional genetic and epi-
genetic mutations during their derivation
(49, 50), although most such mutations can
be neutral. In contrast, germ cells exhibit a
mutation rate that is about 1/10 that of somatic
cells (128, 129), with SSCs bearing a higher
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fidelity for genome replication and oocytes
entering into the meiotic prophase early during
their development (130, 131). Nonetheless,
studies have shown that offspring of aged
fathers who accumulate more mutations may
be susceptible to certain diseases, including
autism spectrum disorders (130, 131). SSC
clones bearing mutations advantageous for
their expansion can dominate a testicular niche
and produce spermatozoa with the same mu-
tations, resulting in offspring with the corre-
sponding disorders (132, 133). Human iPSC
(hiPSC)–derived gametes, which are not orig-
inally protected by the germline mechanisms,
could bear more mutations than in these ex-
amples. A potential strategy to circumvent this
problem may be to reserve embryonic or neo-
natal cells, such as cord blood cells, which
would accumulate fewer mutations, as donors
for future hiPSC generation. Clearly, more
studies are needed to assess the impact of
genetic and epigenetic mutations on human
physiology and diseases.
With the exception of the germ cell origins,
IVG seeks to recapitulate all key genetic and
epigenetic events during germ cell development
in vivo, including epigenetic reprogramming
and programming and meiotic recombination.
In the future, when all the technological con-
cerns have been resolved, it will be crucial to
hold wide discussions about whether to use
IVG-derived gametes for human reproduc-
tion, because such an application involves
somatic cells as human origins and changes
our understanding of the continuity of life
through germ cells.
Conclusions
The salient functions of germ cells are to pre-
serve genetic and epigenetic information with
high fidelity and also, somewhat paradoxically,
to create diversity in this information through
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RES EARCH | R E V I E W
distinctive mechanisms as a driving force for
evolution. With proof-of-concept studies in
mice and a growing extension to humans, IVG
has great potential for future applications.
On the other hand, the genetic as well as epi-
genetic integrities of gametes and developing
germ cells generated through IVG still appear
substantially lower than those of gametes and
germ cells generated under in vivo contexts,
indicating that there is much to be learned
from investigations into germ cell development
in vivo in diverse organisms.
RE FE RENCES AND N OT ES
1. M. Saitou, H. Miyauchi, Gametogenesis from pluripotent stem
cells. Cell Stem Cell 18, 721–735 (2016). doi: 10.1016/
j.stem.2016.05.001; pmid: 27257761
2. W. W. Tang, T. Kobayashi, N. Irie, S. Dietmann, M. A. Surani,
Specification and epigenetic programming of the human
germ line. Nat. Rev. Genet. 17, 585–600 (2016). doi: 10.1038/
nrg.2016.88; pmid: 27573372
3. H. J. Lee, T. A. Hore, W. Reik, Reprogramming the
methylome: Erasing memory and creating diversity. Cell Stem
Cell 14, 710–719 (2014). doi: 10.1016/j.stem.2014.05.008;
pmid: 24905162
4. C. Spiller, P. Koopman, J. Bowles, Sex determination in the
mammalian germline. Annu. Rev. Genet. 51, 265–285
(2017). doi: 10.1146/annurev-genet-120215-035449;
pmid: 28853925
5. L. Wen, F. Tang, Human germline cell development: from the
perspective of single-cell sequencing. Mol. Cell 76, 320–328
(2019). doi: 10.1016/j.molcel.2019.08.025; pmid: 31563431
6. M. A. Edson, A. K. Nagaraja, M. M. Matzuk, The mammalian
ovary from genesis to revelation. Endocr. Rev. 30, 624–712
(2009). doi: 10.1210/er.2009-0012; pmid: 19776209
7. E. A. McGee, A. J. Hsueh, Initial and cyclic recruitment of
ovarian follicles. Endocr. Rev. 21, 200–214 (2000).
pmid: 10782364
8. G. Manku, M. Culty, Mammalian gonocyte and spermatogonia
differentiation: Recent advances and remaining challenges.
Reproduction 149, R139–R157 (2015). doi: 10.1530/
REP-14-0431; pmid: 25670871
9. S. Chuma, T. Nakano, piRNA and spermatogenesis in mice.
Philos. Trans. R. Soc. London Ser. B 368, 20110338 (2013).
doi: 10.1098/rstb.2011.0338; pmid: 23166399
10. M. D. Griswold, Spermatogenesis: The commitment to
meiosis. Physiol. Rev. 96, 1–17 (2016). doi: 10.1152/
physrev.00013.2015; pmid: 26537427
11. M. Kanatsu-Shinohara, T. Shinohara, Spermatogonial stem cell
self-renewal and development. Annu. Rev. Cell Dev. Biol. 29,
163–187 (2013). doi: 10.1146/annurev-cellbio-101512-122353;
pmid: 24099084
12. T. Taketo, The role of sex chromosomes in mammalian germ
cell differentiation: Can the germ cells carrying X and
Y chromosomes differentiate into fertile oocytes? Asian J.
Androl. 17, 360–366 (2015). pmid: 25578929
13. P. S. Burgoyne, The role of the mammalian Y chromosome in
spermatogenesis. Development 101 (suppl.), 133–141 (1987).
doi: 10.1242/dev.101.Supplement.133; pmid: 3503711
14. K. A. Lawson et al., Bmp4 is required for the generation of
primordial germ cells in the mouse embryo. Genes Dev. 13,
424–436 (1999). doi: 10.1101/gad.13.4.424; pmid: 10049358
15. M. Saitou, S. C. Barton, M. A. Surani, A molecular programme
for the specification of germ cell fate in mice. Nature 418,
293–300 (2002). doi: 10.1038/nature00927; pmid: 12124616
16. Y. Ohinata et al., Blimp1 is a critical determinant of the
germ cell lineage in mice. Nature 436, 207–213 (2005).
doi: 10.1038/nature03813; pmid: 15937476
17. Y. Ohinata et al., A signaling principle for the specification of
the germ cell lineage in mice. Cell 137, 571–584 (2009).
doi: 10.1016/j.cell.2009.03.014; pmid: 19410550
18. M. Yamaji et al., Critical function of Prdm14 for the
establishment of the germ cell lineage in mice. Nat. Genet.
40, 1016–1022 (2008). doi: 10.1038/ng.186; pmid: 18622394
19. S. Weber et al., Critical function of AP-2 gamma/TCFAP2C in
mouse embryonic germ cell maintenance. Biol. Reprod. 82,
214–223 (2010). doi: 10.1095/biolreprod.109.078717;
pmid: 19776388
Saitou et al., Science 374, eaaz6830 (2021) 1 October 2021
Corrected 19 October 2021. See full text.
20. J. Nichols, A. Smith, Naive and primed pluripotent states.
Cell Stem Cell 4, 487–492 (2009). doi: 10.1016/
j.stem.2009.05.015; pmid: 19497275
21. Q. L. Ying et al., The ground state of embryonic stem cell
self-renewal. Nature 453, 519–523 (2008). doi: 10.1038/
nature06968; pmid: 18497825
22. M. Buehr et al., Capture of authentic embryonic stem cells
from rat blastocysts. Cell 135, 1287–1298 (2008).
doi: 10.1016/j.cell.2008.12.007; pmid: 19109897
23. P. Li et al., Germline competent embryonic stem cells derived
from rat blastocysts. Cell 135, 1299–1310 (2008).
doi: 10.1016/j.cell.2008.12.006; pmid: 19109898
24. T. Boroviak, R. Loos, P. Bertone, A. Smith, J. Nichols, The
ability of inner-cell-mass cells to self-renew as embryonic
stem cells is acquired following epiblast specification.
Nat. Cell Biol. 16, 513–528 (2014). doi: 10.1038/ncb2965;
pmid: 24859004
25. P. J. Tesar et al., New cell lines from mouse epiblast share
defining features with human embryonic stem cells.
Nature 448, 196–199 (2007). doi: 10.1038/nature05972;
pmid: 17597760
26. I. G. Brons et al., Derivation of pluripotent epiblast stem cells
from mammalian embryos. Nature 448, 191–195 (2007).
doi: 10.1038/nature05950; pmid: 17597762
27. Y. Kojima et al., The transcriptional and functional properties
of mouse epiblast stem cells resemble the anterior primitive
streak. Cell Stem Cell 14, 107–120 (2014). doi: 10.1016/
j.stem.2013.09.014; pmid: 24139757
28. K. Hayashi, H. Ohta, K. Kurimoto, S. Aramaki, M. Saitou,
Reconstitution of the mouse germ cell specification
pathway in culture by pluripotent stem cells. Cell 146,
519–532 (2011). doi: 10.1016/j.cell.2011.06.052;
pmid: 21820164
29. K. Hayashi et al., Offspring from oocytes derived from in
vitro primordial germ cell–like cells in mice. Science 338,
971–975 (2012). doi: 10.1126/science.1226889;
pmid: 23042295
30. A. Smith, Formative pluripotency: The executive phase in a
developmental continuum. Development 144, 365–373
(2017). doi: 10.1242/dev.142679; pmid: 28143843
31. K. Kurimoto et al., Quantitative dynamics of chromatin
remodeling during germ cell specification from mouse
embryonic stem cells. Cell Stem Cell 16, 517–532 (2015).
doi: 10.1016/j.stem.2015.03.002; pmid: 25800778
32. Y. Xiang et al., Epigenomic analysis of gastrulation identifies a
unique chromatin state for primed pluripotency. Nat. Genet.
52, 95–105 (2020). doi: 10.1038/s41588-019-0545-1;
pmid: 31844322
33. M. Kinoshita et al., Capture of mouse and human stem cells
with features of formative pluripotency. Cell Stem Cell 28,
453–471.e8 (2021). doi: 10.1016/j.stem.2020.11.005;
pmid: 33271069
34. L. Yu et al., Derivation of intermediate pluripotent stem cells
amenable to primordial germ cell specification. Cell Stem Cell
28, 550–567.e12 (2021). doi: 10.1016/j.stem.2020.11.003;
pmid: 33271070
35. S. Aramaki et al., A mesodermal factor, T, specifies mouse
germ cell fate by directly activating germline determinants.
Dev. Cell 27, 516–529 (2013). doi: 10.1016/
j.devcel.2013.11.001; pmid: 24331926
36. F. Nakaki et al., Induction of mouse germ-cell fate by
transcription factors in vitro. Nature 501, 222–226 (2013).
doi: 10.1038/nature12417; pmid: 23913270
37. K. Murakami et al., NANOG alone induces germ cells in
primed epiblast in vitro by activation of enhancers. Nature
529, 403–407 (2016). doi: 10.1038/nature16480;
pmid: 26751055
38. J. Zhang et al., OTX2 restricts entry to the mouse germline.
Nature 562, 595–599 (2018). doi: 10.1038/s41586-018-0581-5;
pmid: 30283136
39. J. Tischler et al., Metabolic regulation of pluripotency and
germ cell fate through a-ketoglutarate. EMBO J. 38, e100615
(2019). doi: 10.15252/embj.201899518; pmid: 30257965
40. T. Hirota et al., Fertile offspring from sterile sex chromosome
trisomic mice. Science 357, 932–935 (2017). doi: 10.1126/
science.aam9046; pmid: 28818972
41. J. J. Eppig, M. J. O’Brien, Development in vitro of mouse
oocytes from primordial follicles. Biol. Reprod. 54, 197–207
(1996). doi: 10.1095/biolreprod54.1.197; pmid: 8838017
42. K. Hashimoto, M. Noguchi, N. Nakatsuji, Mouse offspring
derived from fetal ovaries or reaggregates which were cultured
and transplanted into adult females. Dev. Growth Differ. 34,
233–238 (1992). doi: 10.1111/j.1440-169X.1992.tb00012.x
43. K. Morohaku et al., Complete in vitro generation of fertile
oocytes from mouse primordial germ cells. Proc. Natl. Acad.
Sci. U.S.A. 113, 9021–9026 (2016). doi: 10.1073/
pnas.1603817113; pmid: 27457928
44. O. Hikabe et al., Reconstitution in vitro of the entire cycle of
the mouse female germ line. Nature 539, 299–303 (2016).
doi: 10.1038/nature20104; pmid: 27750280
45. E. Habibi et al., Whole-genome bisulfite sequencing of
two distinct interconvertible DNA methylomes of mouse
embryonic stem cells. Cell Stem Cell 13, 360–369 (2013).
doi: 10.1016/j.stem.2013.06.002; pmid: 23850244
46. K. Shirane et al., Global landscape and regulatory principles of
DNA methylation reprogramming for germ cell specification by
mouse pluripotent stem cells. Dev. Cell 39, 87–103 (2016).
doi: 10.1016/j.devcel.2016.08.008; pmid: 27642137
47. M. Yagi et al., Derivation of ground-state female ES cells
maintaining gamete-derived DNA methylation. Nature 548,
224–227 (2017). doi: 10.1038/nature23286; pmid: 28746308
48. J. Choi et al., Prolonged Mek1/2 suppression impairs the
developmental potential of embryonic stem cells. Nature
548, 219–223 (2017). doi: 10.1038/nature23274;
pmid: 28746311
49. U. Ben-David, N. Benvenisty, The tumorigenicity of human
embryonic and induced pluripotent stem cells. Nat. Rev. Cancer
11, 268–277 (2011). doi: 10.1038/nrc3034; pmid: 21390058
50. S. Bar, N. Benvenisty, Epigenetic aberrations in human
pluripotent stem cells. EMBO J. 38, e101033 (2019).
doi: 10.15252/embj.2018101033; pmid: 31088843
Downloaded from https://www.science.org on December 05, 2023
51. S. Shimamoto et al., Hypoxia induces the dormant state in
oocytes through expression of Foxo3. Proc. Natl. Acad. Sci.
U.S.A. 116, 12321–12326 (2019). doi: 10.1073/pnas.1817223116;
pmid: 31147464
52. Y. Feng et al., CLARITY reveals dynamics of ovarian
follicular architecture and vasculature in three-dimensions.
Sci. Rep. 7, 44810 (2017). doi: 10.1038/srep44810;
pmid: 28333125
53. G. Nagamatsu, S. Shimamoto, N. Hamazaki, Y. Nishimura,
K. Hayashi, Mechanical stress accompanied with nuclear
rotation is involved in the dormant state of mouse oocytes.
Sci. Adv. 5, eaav9960 (2019). doi: 10.1126/sciadv.aav9960;
pmid: 31249869
54. N. Hamada et al., Germ cell-intrinsic effects of sex
chromosomes on early oocyte differentiation in mice.
PLOS Genet. 16, e1008676 (2020). doi: 10.1371/
journal.pgen.1008676; pmid: 32214314
55. Y. Yamauchi, J. M. Riel, Z. Stoytcheva, M. A. Ward, Two Y
genes can replace the entire Y chromosome for assisted
reproduction in the mouse. Science 343, 69–72 (2014).
doi: 10.1126/science.1242544; pmid: 24263135
56. N. Hamazaki et al., Reconstitution of the oocyte transcriptional
network with transcription factors. Nature 589, 264–269
(2021). doi: 10.1038/s41586-020-3027-9; pmid: 33328630
57. G. A. Dokshin, A. E. Baltus, J. J. Eppig, D. C. Page, Oocyte
differentiation is genetically dissociable from meiosis in mice.
Nat. Genet. 45, 877–883 (2013). doi: 10.1038/ng.2672;
pmid: 23770609
58. Y. Ishikura et al., In vitro derivation and propagation of
spermatogonial stem cell activity from mouse pluripotent
stem cells. Cell Rep. 17, 2789–2804 (2016). doi: 10.1016/
j.celrep.2016.11.026; pmid: 27926879
59. Q. Zhou et al., Complete meiosis from embryonic stem
cell-derived germ cells in vitro. Cell Stem Cell 18, 330–340
(2016). doi: 10.1016/j.stem.2016.01.017; pmid: 26923202
60. H. Ohta et al., In vitro expansion of mouse primordial germ
cell-like cells recapitulates an epigenetic blank slate. EMBO J.
36, 1888–1907 (2017). doi: 10.15252/embj.201695862;
pmid: 28559416
61. H. Miyauchi et al., Bone morphogenetic protein and retinoic
acid synergistically specify female germ-cell fate in mice.
EMBO J. 36, 3100–3119 (2017). doi: 10.15252/
embj.201796875; pmid: 28928204
62. S. I. Nagaoka et al., ZGLP1 is a determinant for the oogenic
fate in mice. Science 367, eaaw4115 (2020). doi: 10.1126/
science.aaw4115; pmid: 32054698
63. N. Vernet et al., Meiosis occurs normally in the fetal ovary of
mice lacking all retinoic acid receptors. Sci. Adv. 6, eaaz1139
(2020). doi: 10.1126/sciadv.aaz1139; pmid: 32917583
64. A. A. Chassot et al., Retinoic acid synthesis by ALDH1A
proteins is dispensable for meiosis initiation in the mouse
fetal ovary. Sci. Adv. 6, eaaz1261 (2020). doi: 10.1126/
sciadv.aaz1261; pmid: 32494737
65. Y. Lin, M. E. Gill, J. Koubova, D. C. Page, Germ cell-intrinsic
and -extrinsic factors govern meiotic initiation in mouse
8 of 9

RES EARCH | R E V I E W
embryos. Science 322, 1685–1687 (2008). doi: 10.1126/
science.1166340; pmid: 19074348
66. J. A. Thomson et al., Embryonic stem cell lines derived from
human blastocysts. Science 282, 1145–1147 (1998).
doi: 10.1126/science.282.5391.1145; pmid: 9804556
67. T. Nakamura et al., A developmental coordinate of
pluripotency among mice, monkeys and humans. Nature 537,
57–62 (2016). doi: 10.1038/nature19096; pmid: 27556940
68. K. Sasaki et al., The germ cell fate of cynomolgus monkeys is
specified in the nascent amnion. Dev. Cell 39, 169–185
(2016). doi: 10.1016/j.devcel.2016.09.007; pmid: 27720607
69. R. C. V. Tyser, E. Mahammadov, S. Nakanoh, L. Vallier,
A. Scialdone, S. Srinivas, A spatially resolved single cell atlas
of human gastrulation. bioRxiv 2020.07.21.213512 [Preprint]
(2020). https://doi.org/10.1101/2020.07.21.213512.
70. T. W. Theunissen et al., Systematic identification of culture
conditions for induction and maintenance of naive human
pluripotency. Cell Stem Cell 15, 471–487 (2014). doi: 10.1016/
j.stem.2014.07.002; pmid: 25090446
71. Y. Takashima et al., Resetting transcription factor control
circuitry toward ground-state pluripotency in human. Cell
158, 1254–1269 (2014). doi: 10.1016/j.cell.2014.08.029;
pmid: 25215486
72. C. Dong et al., Derivation of trophoblast stem cells from naïve
human pluripotent stem cells. eLife 9, e52504 (2020).
doi: 10.7554/eLife.52504; pmid: 32048992
73. J. K. Cinkornpumin et al., Naive human embryonic stem cells
can give rise to cells with a trophoblast-like transcriptome
and methylome. Stem Cell Reports 15, 198–213 (2020).
doi: 10.1016/j.stemcr.2020.06.003; pmid: 32619492
74. L. Yu et al., Blastocyst-like structures generated from human
pluripotent stem cells. Nature 591, 620–626 (2021).
doi: 10.1038/s41586-021-03356-y; pmid: 33731924
75. X. Liu et al., Modelling human blastocysts by reprogramming
fibroblasts into iBlastoids. Nature 591, 627–632 (2021).
doi: 10.1038/s41586-021-03372-y; pmid: 33731926
76. Y. Niu et al., Dissecting primate early post-implantation development
using long-term in vitro embryo culture. Science 366, eaaw5754
(2019). doi: 10.1126/science.aaw5754; pmid: 31672917
77. D. Chen et al., Human primordial germ cells are specified
from lineage-primed progenitors. Cell Rep. 29, 4568–4582.e5
(2019). doi: 10.1016/j.celrep.2019.11.083; pmid: 31875561
78. O. Gafni et al., Derivation of novel human ground state naive
pluripotent stem cells. Nature 504, 282–286 (2013).
doi: 10.1038/nature12745; pmid: 24172903
79. N. Irie et al., SOX17 is a critical specifier of human primordial
germ cell fate. Cell 160, 253–268 (2015). doi: 10.1016/
j.cell.2014.12.013; pmid: 25543152
80. K. Sasaki et al., Robust in vitro induction of human germ cell
fate from pluripotent stem cells. Cell Stem Cell 17, 178–194
(2015). doi: 10.1016/j.stem.2015.06.014; pmid: 26189426
81. C. Yamashiro et al., Generation of human oogonia from
induced pluripotent stem cells in vitro. Science 362, 356–360
(2018). doi: 10.1126/science.aat1674; pmid: 30237246
82. Y. S. Hwang et al., Reconstitution of prospermatogonial
specification in vitro from human induced pluripotent stem
cells. Nat. Commun. 11, 5656 (2020). doi: 10.1038/
s41467-020-19350-3; pmid: 33168808
83. Y. Zheng et al., Controlled modelling of human epiblast and
amnion development using stem cells. Nature 573, 421–425
(2019). doi: 10.1038/s41586-019-1535-2; pmid: 31511693
84. Y. Kojima et al., Evolutionarily distinctive transcriptional and
signaling programs drive human germ cell lineage specification
from pluripotent stem cells. Cell Stem Cell 21, 517–532.e5 (2017).
doi: 10.1016/j.stem.2017.09.005; pmid: 28985527
85. D. Chen et al., The TFAP2C-regulated OCT4 naive enhancer is
involved in human germline formation. Cell Rep. 25, 3591–3602.
e5 (2018). doi: 10.1016/j.celrep.2018.12.011; pmid: 30590035
86. A. Sybirna et al., A critical role of PRDM14 in human primordial
germ cell fate revealed by inducible degrons. Nat. Commun. 11, 1282
(2020). doi: 10.1038/s41467-020-15042-0; pmid: 32152282
87. M. Pierson Smela, A. Sybirna, F. C. K. Wong, M. A. Surani,
Testing the role of SOX15 in human primordial germ cell fate.
Wellcome Open Res. 4, 122 (2019). doi: 10.12688/
wellcomeopenres.15381.1; pmid: 31583280
88. Y. Kojima et al., GATA transcription factors, SOX17 and
TFAP2C, drive the human germ-cell specification program.
Life Sci. Alliance 4, e202000974 (2021). doi: 10.26508/
lsa.202000974; pmid: 33608411
89. E. Sosa et al., Differentiation of primate primordial germ
cell-like cells following transplantation into the adult gonadal
niche. Nat. Commun. 9, 5339 (2018). doi: 10.1038/
s41467-018-07740-7; pmid: 30559363
Saitou et al., Science 374, eaaz6830 (2021) 1 October 2021
Corrected 19 October 2021. See full text.
90. Y. Sakai et al., Induction of the germ cell fate from pluripotent
stem cells in cynomolgus monkeys. Biol. Reprod. 102, 620–638
(2020). doi: 10.1093/biolre/ioz205; pmid: 31724030
91. T. Kobayashi et al., Principles of early human development
and germ cell program from conserved model systems. Nature
546, 416–420 (2017). doi: 10.1038/nature22812; pmid: 28607482
92. S. Yokobayashi et al., Clonal variation of human induced
pluripotent stem cells for induction into the germ cell fate.
Biol. Reprod. 96, 1154–1166 (2017). doi: 10.1093/biolre/
iox038; pmid: 28453617
93. H. Ohta et al., Cyclosporin A and FGF signaling support the
proliferation/survival of mouse primordial germ cell-like cells
in vitro. Biol. Reprod. 104, 344–360 (2021). doi: 10.1093/
biolre/ioaa195; pmid: 33079185
94. Y. Murase et al., Long-term expansion with germline potential
of human primordial germ cell-like cells in vitro. EMBO J. 39,
e104929 (2020). doi: 10.15252/embj.2020104929;
pmid: 32954504
95. S. Seisenberger et al., The dynamics of genome-wide DNA
methylation reprogramming in mouse primordial germ cells.
Mol. Cell 48, 849–862 (2012). doi: 10.1016/j.
molcel.2012.11.001; pmid: 23219530
96. S. Kagiwada, K. Kurimoto, T. Hirota, M. Yamaji, M. Saitou,
Replication-coupled passive DNA demethylation for the
erasure of genome imprints in mice. EMBO J. 32, 340–353
(2013). doi: 10.1038/emboj.2012.331; pmid: 23241950
97. S. Gkountela et al., DNA demethylation dynamics in the
human prenatal germline. Cell 161, 1425–1436 (2015).
doi: 10.1016/j.cell.2015.05.012; pmid: 26004067
98. W. W. Tang et al., A unique gene regulatory network resets the
human germline epigenome for development. Cell 161, 1453–1467
(2015). doi: 10.1016/j.cell.2015.04.053; pmid: 26046444
99. F. Guo et al., The transcriptome and DNA methylome landscapes
of human primordial germ cells. Cell 161, 1437–1452 (2015).
doi: 10.1016/j.cell.2015.05.015; pmid: 26046443
100. L. Li et al., Single-cell RNA-seq analysis maps development of
human germline cells and gonadal niche interactions.
Cell Stem Cell 20, 858–873.e4 (2017). doi: 10.1016/
j.stem.2017.03.007; pmid: 28457750
101. Á. Vértesy et al., Parental haplotype-specific single-cell
transcriptomics reveal incomplete epigenetic reprogramming
in human female germ cells. Nat. Commun. 9, 1873 (2018).
doi: 10.1038/s41467-018-04215-7; pmid: 29760424
102. F. Fang et al., A PAX5-OCT4-PRDM1 developmental switch
specifies human primordial germ cells. Nat. Cell Biol. 20, 655–665
(2018). doi: 10.1038/s41556-018-0094-3; pmid: 29713018
103. J. Guo et al., Single-cell analysis of the developing human
testis reveals somatic niche cell specification and fetal
germline stem cell establishment. Cell Stem Cell 28, 764–778.
e4 (2021). doi: 10.1016/j.stem.2020.12.004; pmid: 33453151
104. T. Chitiashvili et al., Female human primordial germ cells
display X-chromosome dosage compensation despite
the absence of X-inactivation. Nat. Cell Biol. 22, 1436–1446
(2020). doi: 10.1038/s41556-020-00607-4; pmid: 33257808
105. T. G. Baker, A quantitative and cytological study of germ cells
in human ovaries. Proc. R. Soc. London Ser. B 158, 417–433
(1963). doi: 10.1098/rspb.1963.0055; pmid: 14070052
106. T. Fukuda, C. Hedinger, P. Groscurth, Ultrastructure of developing
germ cells in the fetal human testis. Cell Tissue Res. 161, 55–70
(1975). doi: 10.1007/BF00222114; pmid: 1149138
107. S. Mekhoubad et al., Erosion of dosage compensation impacts
human iPSC disease modeling. Cell Stem Cell 10, 595–609
(2012). doi: 10.1016/j.stem.2012.02.014; pmid: 22560080
108. C. Vallot et al., Erosion of X chromosome inactivation
in human pluripotent cells initiates with XACT coating and
depends on a specific heterochromatin landscape. Cell Stem
Cell 16, 533–546 (2015). doi: 10.1016/j.stem.2015.03.016;
pmid: 25921272
109. S. Patel et al., Human embryonic stem cells do not change
their X inactivation status during differentiation. Cell Rep. 18,
54–67 (2017). doi: 10.1016/j.celrep.2016.11.054; pmid: 27989715
110. D. Chen et al., Germline competency of human embryonic
stem cells depends on eomesodermin. Biol. Reprod. 97,
850–861 (2017). doi: 10.1093/biolre/iox138; pmid: 29091993
111. T. Sato et al., In vitro production of functional sperm in
cultured neonatal mouse testes. Nature 471, 504–507 (2011).
doi: 10.1038/nature09850; pmid: 21430778
112. T. Sato et al., In vitro production of fertile sperm from murine
spermatogonial stem cell lines. Nat. Commun. 2, 472 (2011).
doi: 10.1038/ncomms1478; pmid: 21915114
113. K. Kojima et al., Neonatal testis growth recreated in vitro by
two-dimensional organ spreading. Biotechnol. Bioeng. 115,
3030–3041 (2018). doi: 10.1002/bit.26822; pmid: 30144353
114. Y. Ishikura et al., In vitro reconstitution of the whole male
germ-cell development from mouse pluripotent stem cells.
Cell Stem Cell 10.1016/j.stem.2021.08.005 (2021).
115. T. B. Hildebrandt et al., Embryos and embryonic stem cells
from the white rhinoceros. Nat. Commun. 9, 2589 (2018).
doi: 10.1038/s41467-018-04959-2; pmid: 29973581
116. I. Stévant et al., Dissecting cell lineage specification and sex
fate determination in gonadal somatic cells using single-cell
transcriptomics. Cell Rep. 26, 3272–3283.e3 (2019).
doi: 10.1016/j.celrep.2019.02.069; pmid: 30893600
117. K. Sasaki et al., The embryonic ontogeny of the gonadal
somatic cells in mice and monkeys. Cell Rep. 35, 109075
(2021). doi: 10.1016/j.celrep.2021.109075; pmid: 33951437
118. T. Yoshino et al., Generation of ovarian follicles from mouse
pluripotent stem cells. Science 373, eabe0237 (2021).
doi: 10.1126/science.abe0237; pmid: 34437124
119. Z. Liu et al., Generation of macaques with sperm derived
from juvenile monkey testicular xenografts. Cell Res. 26,
139–142 (2016). doi: 10.1038/cr.2015.112; pmid: 26369429
120. A. P. Fayomi et al., Autologous grafting of cryopreserved
prepubertal rhesus testis produces sperm and offspring.
Science 363, 1314–1319 (2019). doi: 10.1126/science.
aav2914; pmid: 30898927
121. C. Ramathal et al., Fate of iPSCs derived from azoospermic
and fertile men following xenotransplantation to murine
seminiferous tubules. Cell Rep. 7, 1284–1297 (2014).
doi: 10.1016/j.celrep.2014.03.067; pmid: 24794432
122. A. Wutz, Haploid mouse embryonic stem cells: Rapid
Downloaded from https://www.science.org on December 05, 2023
genetic screening and germline transmission. Annu. Rev.
Cell Dev. Biol. 30, 705–722 (2014). doi: 10.1146/
annurev-cellbio-100913-012920; pmid: 25288120
123. I. Sagi, N. Benvenisty, Haploidy in humans: An evolutionary
and developmental perspective. Dev. Cell 41, 581–589 (2017).
doi: 10.1016/j.devcel.2017.04.019; pmid: 28633015
124. T. Ishii, M. Saitou, Promoting in vitro gametogenesis research
with a social understanding. Trends Mol. Med. 23, 985–988
(2017). doi: 10.1016/j.molmed.2017.09.006; pmid: 29032005
125. E. Heard, R. A. Martienssen, Transgenerational epigenetic
inheritance: Myths and mechanisms. Cell 157, 95–109
(2014). doi: 10.1016/j.cell.2014.02.045; pmid: 24679529
126. S. Jaiswal et al., Age-related clonal hematopoiesis associated
with adverse outcomes. N. Engl. J. Med. 371, 2488–2498
(2014). doi: 10.1056/NEJMoa1408617; pmid: 25426837
127. A. Yokoyama et al., Age-related remodelling of oesophageal
epithelia by mutated cancer drivers. Nature 565, 312–317
(2019). doi: 10.1038/s41586-018-0811-x; pmid: 30602793
128. B. Milholland et al., Differences between germline and somatic
mutation rates in humans and mice. Nat. Commun. 8, 15183
(2017). doi: 10.1038/ncomms15183; pmid: 28485371
129. L. Ségurel, M. J. Wyman, M. Przeworski, Determinants of
mutation rate variation in the human germline. Annu. Rev.
Genomics Hum. Genet. 15, 47–70 (2014). doi: 10.1146/
annurev-genom-031714-125740; pmid: 25000986
130. A. Kong et al., Rate of de novo mutations and the importance
of father’s age to disease risk. Nature 488, 471–475 (2012).
doi: 10.1038/nature11396; pmid: 22914163
131. J. M. Goldmann et al., Parent-of-origin-specific signatures of
de novo mutations. Nat. Genet. 48, 935–939 (2016).
doi: 10.1038/ng.3597; pmid: 27322544
132. E. Giannoulatou et al., Contributions of intrinsic mutation rate
and selfish selection to levels of de novo HRAS mutations in the
paternal germline. Proc. Natl. Acad. Sci. U.S.A. 110, 20152–20157
(2013). doi: 10.1073/pnas.1311381110; pmid: 24259709
133. G. J. Maher et al., Selfish mutations dysregulating RAS-MAPK
signaling are pervasive in aged human testes. Genome Res. 28,
1779–1790 (2018). doi: 10.1101/gr.239186.118; pmid: 30355600
AC KNOWLED GME NTS
We thank the members of our laboratory for their input on this study.
We apologize to the colleagues whose primary works are not cited
because of space constraints. Funding: This work was supported
by a Grant-in-Aid for Specially Promoted Research from JSPS
(17H06098), a JST-ERATO grant (JPMJER1104), a grant from HFSP
(RGP0057/2018), and grants from the Pythias Fund and Open
Philanthropy Project to M.S., and by a Grant-in-Aid for Scientific
Research (A) from JSPS (17H01395) and a Grant-in-Aid for Scientific
Research on Innovative Areas from JSPS (18H05545) to K.H.
Competing interests: M.S. and K.H. are inventors on patent
applications relating to induction of germ cells from pluripotent stem
cells filed by Kyoto University and Kyushu University, respectively, and
are founders of Houjou, Inc.
10.1126/science.aaz6830
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 Mammalian in vitro gametogenesis
Mitinori Saitou and Katsuhiko Hayashi

Science 374 (6563), eaaz6830. DOI: 10.1126/science.aaz6830

Reconstituting reproduction in culture

Research on in vitro gametogenesis (IVG) aims to reconstitute germ cell development, oogenesis and
spermatogenesis, in culture. Saitou and Hayashi review some of the recent developments in mammalian IVG.
Advances in methods and culture conditions in mice to generate mature oocytes and spermatocytes from pluripotent
stem cells have informed similar studies with nonhuman primate and human cells, but differences among species
are clear. IVG has great potential for reproductive medicine, including novel diagnosis and modeling of infertility. The

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realization of human IVG requires further intensive efforts, but as technical hurtles are overcome, careful consideration
must be given to the potential application of methods for reproductive purposes. —BAP

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