Scientific and Clinical Advances Advisory
Committee Paper
Paper Title:
Update on in vitro derived gametes
Paper Number:
SCAAC (02/11)04
Meeting Date:
2 February 2011 (circulated November 2010,
members requested further discussion)
Agenda Item:
9
Author:
Rachel Fowler
For information or
decision?
Decision
Resource Implications:
None
Implementation
None
Communication
The Authority will be informed if SCAAC’s view on
this issue changes.
Organisational Risk
Low
Members are asked to:
 note this update on research into in vitro
derived gamete
Recommendation to the
Committee:
 advise the Executive if they are aware of
other advances in research of in vitro
derived gametes
 advise the Executive whether their view of
September 2009 has changed.
Evaluation
None
Annexes
None
TRIM reference: 2010/06896
Committee:
SCAAC
Doc title: 2011-02-02 SCAAC paper – Update on in vitro
derived gametes
Page 1 of 8
Paper Number SCAAC(02/11)04
1.
Lay summary
1.1.
Human germ cells (sperm and eggs) are derived from a type of stem cell
called primordial germ cells. They are derived by the process of
gametogenesis in the testes and ovaries of men and women. Researchers
are investigating whether it is possible to carry out gametogenesis in the
laboratory using primordial germ cells, embryonic stem cells or other
human cells. Sperm and eggs derived from such cells in the laboratory are
called in vitro derived gametes.
1.2.
In vitro derived gametes can be used for research purposes, eg, research
into germ cell development and cell differentiation. In vitro derived
gametes could also be used in treatment. For people who are unable to
produce their own eggs or sperm, in vitro derived gametes are a potential
method by which they could have children that are genetically related to
them. It is important to note that the HFE Act 1990 (as amended) prohibits
the use of in vitro derived gametes for treatment purposes.
2.
Introduction
2.1.
An HFEA research licence would be required by researchers in the UK if
they wished to investigate whether human sperm and eggs derived in vitro
could undergo fertilisation and the early stages of embryo development. It
is therefore important that the HFEA is aware of progress into research on
in vitro derived gametes.
2.2.
The Committee last reviewed research on in vitro derived human gametes
in September 2009. It was the Committee’s view that one of the main
hindrances to in vitro derived gametes was incorrect imprinting. It was
suggested that transplanting gamete precursor cells to their normal
environment for the later stages of gamete maturation could help resolve
this. However, the transplantation of human gamete precursor cells
(derived in vitro) into was not at the time a viable, safe approach. Despite
progress, no researched published at the time convincingly showed that
human embryonic stem cells could be differentiated in vitro into mature
human sperm.
3.
Research
Male gametes
3.1.
Mouse embryonic stem cells (ES cells) can differentiate within an
embryoid body to form haploid primordial germ cells (PG cells) which,
once injected into eggs, can form blastocyst-like structures. The derivation
of PG cells through this method can be greatly enhanced when co-
TRIM reference: 2010/06896
Committee:
SCAAC
Doc title: 2011-02-02 SCAAC paper – Update on in vitro
derived gametes
Page 2 of 8
Paper Number SCAAC(02/11)04
cultured with somatic cells expressing BMP41. Furthermore, seminiferous
tubules can form when the cells are transplanted into the testicular
environment. The isolation of haploid cells from embryoid bodies which,
although not resembling spermatozoa, can also be injected into oocytes
and form blastocyst-like structures2. Further embryonic development,
however, has not been explored and in a study with similar findings, the
derivation process was relatively ill-defined and inefficient3.
3.2.
The technique of using embryoid bodies to derive gametes in vitro has
also been explored with human, rather than mouse, ES cells. However,
the full competence of any resulting haploid or gamete-like cells has not
been demonstrated4,5. Other work on deriving PG cells from ES cells has
shown an improved rate of derivation by culturing with human fetal
gonadal stromal cells6.
3.3.
Using a second method, monolayer culture in mice, ES cells are capable
of differentiating into male germ cells 7. Two spermatogonal stem cell lines
have been established and cells were able to undergo spermatogenesis
and acquired a sperm-like morphology. Once injected into eggs and
transferred to a female mouse, pregnancies were established and live
offspring born. The proportion of embryos transferred that gave rise to a
live birth, however, was low and all progeny died within months of birth.
Male germ cells can also be derived from bone marrow derived
mesynchymal stem cells in the mouse8.
3.4.
Further study is needed of the processes involved in derivation before fully
normal sperm function, subsequent egg fertilisation and embryo
development are seen. Also, directed differentiation may be favourable to
allowing random differentiation and then selection of rare cells that
1
Toyooka Y et al (2003) embryonic stem cells can form germ cells in vitro. Proc Natl
Acad Sci USA 100:11457-11462
2
Geijsen N et al (2004) Derivation of embryonic germ cells and male gametes from
embryonic stem cells. Nature 427:148-154
3
Aflatoonian B et al (2009) In vitro post-meiotic germ cell development form human
embryonic stem cells. Human Reproduction 24(12): 3150-3159
4
Clark AT et al (2004) Spontaneous differentiation of germ cells from human embryonic
stem cells in vitro. Hum Mol Genet 13: 727-739
5
Kee K et al (2009) Human DAZL, DAZ and BOULE genes modulate primordial germ
cell and haploid gamete formation. Nature 462:222-225
6
Park et al (2009) Derivation of primordial germ cells from human embryonic and induced
pluripotent stem cells is significantly improved by the co-culture with human fetal gonadal
cells. Stem Cells 27(4):783-795
7
Nayernia K et al (2006) In vitro differentiated embryonic stem cells give rise to male
gametes that can generate offspring mice. Dev Cell 11: 125-132
8
Nayernia K et al (2006) Derivation of male germ cells from bone marrow stem cells.
Lab Invest 86: 654-663
TRIM reference: 2010/06896
Committee:
SCAAC
Doc title: 2011-02-02 SCAAC paper – Update on in vitro
derived gametes
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Paper Number SCAAC(02/11)04
express some germ cell characteristics, as seen with the embryoid body
methodology.
3.5.
It has been demonstrated that multipotent cells exist in the postnatal
testes which have varied ability to form PG cells. Multipotent germ cell
lines have been generated from neonatal mouse testes via culture induced
reprogramming9,10. The cell lines created in this research resembled ES
cells but of the two, only one maintained spermatogonial stem cell
function. Further research has shown the possibility of generating these
multipotent germ cell lines from mouse testes without reprogramming
growth factors11.
3.6.
In vitro differentiation of PGC-like cells from the epiblast of mice has been
achieved, by directing the differentiation of mouse epiblasts into PGC-like
cells (EpiPGC) 12. Spermatogenesis, fertilisation and subsequent
embryogenesis were observed, leading to live births. The efficiency of cell
induction was found to increase with the presence of BMP4 (acting via
induction of Blimp1 and Prdm14) but the overall rate was still low. With
regards to epigenetics, the EpiPGCs were able to undergo imprinting
erasure, although not entirely.
3.7.
Further work is required on the signalling pathways involved in
differentiation into the PG cell state, the nature of the epiblast cells for
germ cell fate and the epigenetic reprogramming in PG cells after their
specification13. PGC-like cells have also been induced from human ES
cells and from mesenchymal stem cells in the mouse14 and human15.
Female gametes
3.8.
Embryonic stem cells (ES cells), extracted from the inner cell mass of a
blastocyst, are pluripotent and evidence suggests their potential to
differentiate in vitro into germ cells and female gametes.
9
Knatsu-shinohara et al (2004) Generation of pluripotent stem cells from neonatal mouse
testis. Cell 119:1001-1012
10
Seandel M et al (2007) Generation of functional multipotent adult stem cells from
GPR125+ germline progenitors. Nature 449:346-350
11
Guan K et al (2006) Pluripotency of Spermatogonial stem cells from adult mouse
testis. Nature 440:1199-1203
12
Ohinata Y et al (2009) A signalling principle for the specification of the germ cell
lineage in mice. Cell 137:571-584
13
Saitou M et al (2010) Germ cell specification in mice: signalling, transcription,
regulation, and epigenetic consequences. Reproduction 139:931-942
14
Nayernia (2006) Derivation of male germ cells from bone marrow stem cells. Lab Invest
86(7):654-63
15
Drusenheimer (2007) Putative human male germ cells from bone marrow stem cells.
Soc Reprod Fertil Suppl 63:69-76
TRIM reference: 2010/06896
Committee:
SCAAC
Doc title: 2011-02-02 SCAAC paper – Update on in vitro
derived gametes
Page 4 of 8
Paper Number SCAAC(02/11)04
3.9.
The in vitro culture of mouse ES cells has shown their ability to
differentiate into oocyte-like cells with follicle-like structures surrounding
them. Subsequent parthenogenetic activation of the oocytes has given rise
to blastocysts16.
3.10.
Embryoid bodies have provided further evidence of ES cell capability to
differentiate down the female germ cell pathway, including into ovarian
structures containing putative oocytes. Prolonged culturing of these
putative oocytes revealed their expression of oocyte-specific markers but
their stage of development and whether they could be fully functional has
not been investigated17.
3.11.
The differentiation of ES cells within embryoid bodies has also been
researched and shown granulosa cells to be capable of inducing ESderived PG cells into oocyte-like cells18. The generation of PG cells from
ES cells has also been reported and the gene expression pattern of the
resulting PG cells suggested they were similar to primordial germ cells
created in vivo and by co-culture with hamster ovary cells some entered
into meiosis19.
3.12.
Although there is evidence of the potential for ES cells to differentiate in
vitro into female gametes, the methods are currently inefficient and the
gametes’ functionality is not fully known. Increasing the efficiency of
deriving female gametes from mouse ES cells has been investigated
through using the embryoid body and monolayer culture methods20. The
addition of LIF (Leukemia inhibitory factor) to culture medium has been
found to increase the number of oocyte-like cells and oocyte specific gene
expression.
3.13.
Evidence exists that the female primordial follicle pool is replenished
during postnatal life of adult mammals. The primordial follicle pool number
in mice is stable during the first 100 days of life, despite the loss of many
primordial follicles21, 22 and that adult mice replenish their primordial follicle
16
Hubner K et al (2003) Derivation of oocytes from mouse embryonic stem cells.
Science 300:1251-1256
17
Lacham-Kaplan O et al (2006) Testicular cell conditioned medium supports
differentiation of embryonic stem cells into ovarian structures containing oocytes. Stem
Cells 24: 266-273
18
Qing T et al (2007) Induction of oocyte-like cells from mouse embryonic stem cells by
co-culture with ovarian granulosa cells. Differentiation 75:3902-911
19
Eguizabal C et al (2009) Generation of primordial germ cells from pluripotent stem
cells. Differentiation 78(2-3):116-123
20
Salvador LM et al (2008) The promoter of the oocyte-specific gene, Gdf9, is active in
population of cultured mouse embryonic stem cells with an oocyte-like phenotype.
Methods 45:172-81
21
Johnson J et al (2004) Germline stem cells and follicular renewal in the postnatal
mammalian ovary. Nature 428: 145-150
TRIM reference: 2010/06896
Committee:
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Doc title: 2011-02-02 SCAAC paper – Update on in vitro
derived gametes
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pool during days 4-5 of each reproductive cycle 23,24.
3.14.
The primordial follicle pool is also regenerated in adult mice after exposure
to doxorubicin (an inducer of oocyte apoptosis)25 and can be increased by
injection of class I/II histone deactelyase (HDAC) inhibitor, trichostatin-A26.
HDAC is suggested to repress the promoter of Stra8, also targeted by
retinoic acid (RA), which encodes a germline-specific protein required for
meiotic competency in germ cells. As such it contributes, with RA, to
determining whether germ cells enter meiosis27.
3.15.
Evidence also comes from two mutant mice lines deficient in the enzyme
CASPASE6 or Cables1-null. Cables1 encodes a cell cycle regulatory
protein involved in constraining the rate of oocyte renewal in adult ovaries.
Female adult mice of the Cables1-null line have increased oocyte
generation, but a decrease in oocyte quality28.
3.16.
There have been attempts to isolate germ stem cells (GS cells) that
possibly exist in female adult ovaries. In particular, ovarian surface
epithelium (OSE) cells have been investigated and mitotically active germ
cells expressing the germ cell specific VASA protein have been reported in
or near to the OSE of juvenile and adult mice29.
3.17.
Research in the human has reported the culture of OSE cells from human
adults and the derivation from these of follicle cells and cells with the
oocyte phenotype30. Human cells with pluripotent ES cell characteristics
have been reported to be found amongst OSE cells extracted from adult
ovaries. These cells have been shown to develop into oocyte-like cells and
some have spontaneously developed into blastocyst-like structures,
22
Kerr JB et al (2006) Quantification of healthy molecules in the postnatal and adult
mouse ovary: evidence for maintenance of primordial follicle supply. Reproduction 132:
95-109
23
Allen E (1923) Ovogenesis during sexual maturity. Am J Anat 31:439-482
24
Johnson J et al (2005) Oocyte generation in adult mammalian ovaries by putative germ
cells derived from bone marrow and peripheral blood. Cell 122:303-315
25
Borovskaya TG et al (2006) Morphological state of rat ovaries in the early and late
periods after injection of vespid. Bull Exp Biol Med 141:645-657
26
Johnson J et al (2005) Oocyte generation in adult mammalian ovaries by putative germ
cells derived from bone marrow and peripheral blood. Cell 122:303-315
27
Wang N and Tilly JL (2010) Epigentic status determines germ cell meiotic commitment
in embryonic and postnatal mammalian gonads. Cell 9(2):339-349
28
Lee H-L et al (2007) Loss of CABLES-1, a cyclin dependent kinase-interacting protein
that inhibits cell cycle progression, results in germline expansion at he expense of oocyte
quality in adult female mice. Cell 6:2678-2684
29
Johnson J et al (2004) Germline stem cells and follicular renewal in the postnatal
mammalian ovary. Nature 428:145-150
30
Bukovsky A et al (2005) Oogenesis in cultures derived from adult human ovaries.
Reprod Biol Endocrinol 3:17-25
TRIM reference: 2010/06896
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Doc title: 2011-02-02 SCAAC paper – Update on in vitro
derived gametes
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Paper Number SCAAC(02/11)04
possibly resembling a parthenogenetic embryo31.
3.18.
In the mouse it has been demonstrated in that proliferative germ cells can
be purified from neonatal or adult mouse ovaries, maintained in vitro,
transplanted into sterilised mouse ovaries and give rise to oocytes which
can be fertilised and produce viable offspring32. This evidence supports
the view that there remains a small population of PG cells or similar in the
postnatal and adult ovary which could be used for in vitro derivation of
eggs.
3.19.
Researchers in the field have also investigated the possibility of isolating
PG cells from outside the gonads. Evidence that PG cells are present in
bone marrow and peripheral blood cells has been reported and
transplantation of these into the ovaries of sterilised mice restarts
oogenesis in the ovaries33, although competency of the oocytes has not
yet been established.
4.
Timescale for introduction
4.1.
Members of SCAAC and the HFEA Horizon Scanning Panel have
previously estimated a timescale for deriving gametes in vitro of between
five and ten years, with a shorter timescale for deriving sperm than eggs.
4.2.
In 2008 the Hinxton Group published a review of research into in vitro
derived gametes from pluripotent stem cells34 and estimated that it would
be more than ten years before such gametes were likely to be developed.
The group suggested that in vitro derived gametes would not be available
for treatment purposes until several years later and that creation of
oocytes from cells carrying the X and Y chromosomes and sperm from
cells carrying two copies of the X chromosome would be very difficult.
5.
Regulation
5.1.
Under the HFE Act 1990 (as amended) in vitro derived gametes are not
‘permitted gametes’ and therefore cannot be used for treatment purposes.
Permitted gametes are defined as eggs or sperm which have been
produced by or extracted from the ovaries of a woman or testes of a man,
and whose nuclear or mitochondrial DNA has not been altered.
31
Virant-Klun I et al (2009) Parthenogenetic embryo-like structures in the human ovarian
surface epithelium cell culture in postmenopausal women with no naturally present
follicles and oocytes. Stem cells Dev 18:137-150
32
Zou et al (2009) Production of offspring form a germline stem cell line derived from
neonatal ovaries. Nat Cell Biol 11:631-636
33
Johnson J et al (2005) Oocyte generation in adult mammalian ovaries by putative germ
cells derived from bone marrow and peripheral blood. Cell 122:303-315
34
Mathews et al (2009) Pluripotent stem cell-derived gametes: truth and (potential)
consequences. Cell stem cell 5(1): 11-14
TRIM reference: 2010/06896
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derived gametes
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Paper Number SCAAC(02/11)04
5.2.
Gametes can, however, be derived in vitro and used for research
purposes. This can be done without a licence from the HFEA, unless
researchers wished to use in vitro derived gametes to create an embryo to
test whether they are capable of fertilisation.
6.
Risks
6.1.
The HFEA Horizon Scanning Panel in June 2009 and the Hinxton Group
in their 2008 report both gave the view that the process of deriving
gametes in vitro was very complex and the safety issues were largely
unknown. Further knowledge was thought to be required around
gametogenesis and in particular epigenetics.
6.2.
It was the Panel’s view that animal models, investigation of the meiotic
process and long term studies of offspring resulting from in vitro derived
gametes were required before the technique could be considered
successful or potentially used in treatment. It was similarly the view of the
Hinxton Group that it would be critical to assess the quality of in vitro
derived gametes.
6.3.
In September 2009, members of SCAAC expressed the opinion that
although research into in vitro derivation of sperm had progressed that
year there was no published literature at the time which convincingly
showed human ES cells could be differentiated into mature sperm. The
Committee suggested the main hindrance to in vitro derivation of gametes
was the erasure and then re-imprinting of the genome, and that later
stages of derivation could be aided by returning the immature gametes to
their normal environment.
7.
Conclusion
7.1.
Whilst progress has been made in deriving gametes in vitro problems
remain, further work is required to establish a directed and efficient
method of inducing germ cells from embryonic, pluripotent and germ stem
cells. Secondly, further research is required before it can be demonstrated
that the in vitro derived germ cells have full functionality, as seen with in
vivo germ cells. This would include further work to discover the epigenetic
status of the derived germ cells and ensure that it was correct. Thirdly,
research is required on in vitro derivation of gametes in mammals other
than mice, as the understanding of the process in other mammals,
including humans, is less advanced.
7.2.
Taking into account the summary of the literature, members are asked to
advise the Executive if they are aware of other advances in research of
artificially derived gametes and whether their view of September 2009,
summarised in para 2.2, has changed.
TRIM reference: 2010/06896
Committee:
SCAAC
Doc title: 2011-02-02 SCAAC paper – Update on in vitro
derived gametes
Page 8 of 8