Transcript MAKING GAMETES AND HELPING EMBRYOS

MAKING GAMETES FROM STEM
CELLS
Anna Veiga1,2, Cristina Eguizabal1
1.- Centre de Medicina Regenerativa de
Barcelona
2.- Institut Universitari Dexeus. Barcelona
Pluripotent Stem Cells (PSC)
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Gamete differentiation from PSC(ES/iPS)
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Derivation of Primordial Germ Cells (PGCs)
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Oocyte derivation (mouse/human)
Sperm derivation (mouse/human)
Oogenesis in the adult female
 Pluripotent Stem Cells can be obtained
from cells located in the inner cell mass
of blastocysts (hESC) and from nuclear
reprogramming (SCNT and iPS)
Hochedlinger, Development 2009 (adapted
from Waddington, 1957)
Science,1998
Science, 2007
Cell. 2007 I
Induction of pluripotent stem cells from adult
human fibroblasts by defined factors.
Takahashi K, Tanabe K, Ohnuki M, Narita M,
Ichisaka T, Tomoda K, Yamanaka S.
Derivation of PGCs from PSCs
2008
• Generation a novel protocol based on the expression of CXCR4 receptor during the in
vitro differentiation of PGCs from hES.
• This differentiation process is accompanied by the development of putative Sertoli-like
cells.
2008
• Generation a novel protocol (EBs + BMP4) for differentiating in vitro PGCs from hES.
• These PGCs expressed specific germ cell markers and showed a removal of the
parental imprints and chromating modification changes.
2009
• The generation and characterization of hESC lines stably carrying a VASApEGFP-1 reporter construct that expresses GFP in a population of differentiating
hESC that show expression of characteristic markers of primordial germ cells.
2009
• Pluripotent stem cells (both hESC and iPS) give rise to in vitro PGC
following 7 days of differentiation on hFGC
• They correspond to immature PGCs (developmental stage in vivo between
specification and less than 9 week of gestation)
• Initiation of imprinting erasure is dependent on the epigenetic status of the
pluripotent stem cell from which iPGCs are generated.
Oocyte derivation
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Formation of follicular structures
After 2 weeks in culture,
structures similar to
advanced follicle-like
structures appear. Most of
them degenerate but
around 20% give rise to
“oocytes”> 40 µm.
• The functional activity of the
follicles is evidenced by
estradiol production
Oocyte-like structures
derived from ES cells
• At D26 of culture, oocyte-like
cells ( 50-70 µm) are
released from surrounding
somatic cells and float freely
in the supernatant.
• All ZP markers are + except
ZP1 (accounting for fragile
ZP
structure)
• Addition of gonadotrophins
to isolated follicles results in
extrusion of “oocytes” and
formation of a polar body-like
structure
Blastocyst-like structures
derived from ES cells
• At about day 43, structures similar (morphology
and markers) to preimplantation embryos are
found.
These blastocyst-like structures
represent parthenotes (probably)
induced by culture conditions
Differentiation, 2007
• Granulosa cells are
effective in inducing the
differentiation of ES
derived PGCs into
oocyte-like cells through
direct cell-to-cell
contacts
• Novel in vitro system to
study oogenesis
Nature Cell Biology 2006
•Stem cells isolated from the skin of porcine
fetuses have the intrinsic ability to differentiate
into oocyte-like cells.
• When differentiation was induced, a
subpopulation of these cells expressed
markers (Oct4, GDF9b,DAZL and Vasa),
consistent with germ-cell formation.
•On further differentiation, these cells formed
follicle-like aggregates that secreted
oestradiol and progesterone and responded to
gonadotropin stimulation.
• Some of these aggregates extruded
large oocyte-like cells that expressed
oocyte markers, such as ZP, and
meiosis markers,(SCP3).
• Some of these oocyte-like cells
spontaneously developed into
parthenogenetic embryo-like
structures.
2006
• Differentiation protocol using testicular cell conditioned media for derivating
in vitro putative ·oocytes” from hES.
• These oocytes expressed some oocyte-specific markers.
Sperm derivation
NATURE,
2004
• Culture conditions: Embryoid body
differentiation
• Marker expression
• pluripotency (Oct4).
• GC development markers (Stella ,Fragilis)
• Genes exclusively expressed in GC line
(Dazl, Piwil2, Rnf17, Rnh2, Tdrd1 y
Tex14)
During embryoid body formation
pluripotency markers expression
decreased with some expression of GC
specific genes
PGC differentiation to functional gametes
•Up-regulation of genes associated with male
germ cell development (Sry, Acrosin, Haprin,
markers of Sertoli cells)
•No expression of Zp proteins.
•Within the context of EB differentiation, the
default programm of female gametogenesis is
suppressed.
Meiosis
EB microenvironment is permissive for male germ
cell development and meiotic maturation, even
though highly inefficient
Sperm morphology
• EB derived cells
have a similar
morphology to testis
derived haploid cells.
Round spermatids?
Biological functionality
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ICSI with haploid cells into recipient
oocytes
50% of the embryos cleaved to 2 cell stage
20% reach the blastocyst stage.
FISH: normal for sex chromosomes
PNAS, 2003
• Germ cell in vivo production from mESC.
• Transplantation of ESC aggregates to testicular
capsule.
• ESC derived cells can participate in spermatogenesis
and produce functional germ cells in vitro
Nayernia et al, Developmental Cell 2006
• Establishment of two cell lines (Spermatogonial stem cellsSSCs) with patterns of differentiation towards male germ
cells .
• Derived from mESC (directed gene expression for
premeiotic and haploid male germ cells)
• Motility of cells
• Formation of tail-like structures
• Positive for PGCs, premeiotic
(Oct 4, Fragilis, Stella,..),,
meiotic (Scp3, Acr) and
postmeiotic male germ cells
markers
• Formation of sperm
structures.
• Acrosome like
structures
• Condensation of
the nucleus
• ICSI of in vitro-generated cells (haploid
cells) into oocytes of wildtype females.
• Polar body extrusion, PN formation and
normal features of embryo development.
• Transfer into the oviducts of
pseudopregnant females
• 65 embryos transferred
• 12 animals born.
• Larger or smaller offspring
• Premature death (5 days to 5
months after birth).
• Abnormal methylation patterns
and phenotypic abnormalities
interference with the essential reprograming events in
gametogenesis
• BM stem cells are able to
transdifferentiate into male
germ cells that express
molecular markers of PGC
(fragilis, stella,Mvh..)
• Expression of spermatogonial
stem cells and spermatogonia
markers (CKIt, DAZL..)
Hum Reprod, 2009
• hESC can form PGC and post
meiotic spermatids in vitro.
• Gene expression profiles
characteristics of oocyte development
and follicle like structures were
detected. No oocyte with ZP was
recognised.
• hESC create a favourable niche for
spermatogenesis?
Nature, 2009
• Generation of in vitro PGC from male and
female hESC
• Silencing and overexpression of genes
encoding germ-cell specific cytoplasmic
RNA binding proteins
• The human gene DAZL functions in PGC
formation
• DAZ and BOULE promote germ-cell
progression to meiosis and formation of
haploid germ cells that ressemble round
spermatids
2011
• iPS cells can form in vitro meiotic and postmeiotic haploid cells over-expressing DAZ,
DAZL and BOULE.
2011
• Generation of a novel
differentiating 2 stepprotocol without
overexpressing any
genes.
•iPS can form in vitro
meiotic and haploid likecells.
•The post-meiotic-like
cells generated have
features of human
spermatids expressing
Acrosin.
2011
• Generation of in vitro PGC-like cells from
mES and miPS through epiblast-like cells
having a normal global transcription profiles,
epigenetic reprogramming,and cellular
dynamics.
• These PGCLCs express Integrin-b3 and
SSEA1 markers of spermatogonias.
• Healthy offspring with normal methylation
patterns of imprinted genes.
Oogenesis in the adult female?
Nature 2004
• Juvenile and adult mouse ovaries
possess mitotically active germ
cells that, based on rates of oocyte
degenetration are needed to
replenish the oocyte pool.
• Presence of cells expressing
meiotic markers in juvenile and
adult mouse ovaries
• Wild type ovaries grafted into
transgenic mice (GFP) become
infiltrated with GFP positive cells
that form follicles
Cell, 2005
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Rapid generation of 100s of oocytes
Extragonadal source of germ cells?
Germline markers in BM
BM transplantation restores oocyte
production in wild type mice sterilized by
chemotherapy
• Donor derived oocytes observed in females
following peripheral blood transplantation
BM is a potential source of GC that
sustain oocyte production in
adulthood
Nature,2006
• Bone marrow cells do
not give rise to
oocytes and do not
enhance ovulation of
endogenous oocytes
in transplanted mice
Transplantation and parabiotic
mouse model.
Capacity of circulating BM to
generate ovulated oocytes?
• Circulating cells do not give rise to
mature ocytes in long term parabionts
• Circulating cells do not give rise to
mature oocytes in injured parabionts
Conclusions
• PGCs have been obtained both from hESC and iPS cells
• Oocyte-like cells can be obtained from embryonic and fetal
stem cells.
• Male germ cells and functional sperm have been obtained
through ESC
• Offspring generated from ESC derived sperm are highly
abnormal
• The use of such gametes in ART remains a “distant
prospect"
• Stem cell derived gametes can become a valuable
resource for research: germ cell development , epigenetic
reprogramming and germline gene modification
• Oogenesis does not take place in the adult human female
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