Totipotency
Cell loss during preimplantation development
Hilde Van de Velde, PhD
Tours, April 11, 2008
Learning objectives
z
Definitions
J Totipotency
J Plasticity
z
Cell loss during preimplantation development
J Fragmentation
J Cryodamage
J Biopsy for PGD/PGS
2
Tours, April 11, 2008
The totipotent cell
is able to develop into
normal fertile offspring
3
Tours, April 11, 2008
1
The totipotent cell in the blastocyst (day 6)
ICM
ICM
TE
foetus
trophoblast
4
Tours, April 11, 2008
The invading blastocyst (day 8)
ICM → bilaminar germ disc: epiblast and hypoblast
Epiblast Hypoblast
5
Tours, April 11, 2008
The embedded blastocyst (day 15)
ICM → → trilaminar germ disc: gastrulation
Extra embryonic
Endoderm: yolk
Ectoderm: amnion
Mesoderm: cord
Epiblast
Hypoblast
Embryonic
Endoderm
Ectoderm
Mesoderm
PGC
Germ layers
Trophoblast
6
Tours, April 11, 2008
2
Pluripotent human embryonic stem cells
Trophoblast
ICM
ESC
Thomson Science 1998,282:1145-1147
Reubinoff Nature Biotechnology 2000,18:399-404
Extra embryonic
Endoderm
Ectoderm
Mesoderm
Embryonic
Endoderm
Ectoderm
Mesoderm
PGC
Germ layers
Xu Nature 2002,20:1261-1264
Hyslop STEM CELLS 2005,23:1035-1043
7
Tours, April 11, 2008
Pluripotent human embryonic stem cells
Trophoblast
ICM
ESC
Pluripotent
Thomson Science 1998,282:1145-1147
Reubinoff Nature Biotechnology 2000,18:399-404
Extra embryonic
Endoderm
Ectoderm
Mesoderm
Embryonic
Endoderm
Ectoderm
Mesoderm
PGC
Germ layers
Xu Nature 2002,20:1261-1264
Hyslop STEM CELS 2005,23:1035-1043
Legally: no hybrids
8
Tours, April 11, 2008
Expression of ‘stemness’ markers
in human preimplantation embryos and ESC
Cauffman Greet
POU5F1_iA
SOX2
SALL4
NANOG
9
+
+
+
+
Tours, April 11, 2008
3
Expression of ‘stemness’ markers
in human preimplantation embryos and ESC
Cauffman Greet
TE
+
+→+
-
-
POU5F1_iA
SOX2
SALL4
NANOG
ICM
+
+
+
+
+
+
+
+
The zygote does not express ‘stemness’ markers
10
Tours, April 11, 2008
Embryonic genome switch
maternal control
day 0
16 hours
22 hours
embryonic control
day 2
day 3
day 4
day 5
Braude Nature 1988,13:1461-1470
Monk Oncogene 2001, 20, 8085-8091
Dobson Hum Mol Genet 2004, 13:1461-1470
Cauffman Mol Hum Reprod 2005,11:173-181
Cauffman Mol Hum Reprod 2005,11:405-411
11
Tours, April 11, 2008
NANOG expression in human preimplantation embryos
NANOG expression in some ICM cells?
J = totipotent cells
J = good quality ICM cells
J = ICM cells from which hESC can be derived
J = cells committed to epiblast
J = future primordial germ cells
Are ESC an in vitro artefact?
12
Tours, April 11, 2008
4
Totipotency
z
Conclusions
J The zygote is the ultimate human totipotent cell
J There is no marker for totipotency, there are ‘stemness’ markers
J NANOG is the best marker for ICM cells
z
Questions
J What is an ESC? What is the origin of ESC?
J Where is the totipotent cell in the embryo?
13
Tours, April 11, 2008
Learning objectives
z
Definitions
J Totipotency
J Plasticity
z
Cell loss during preimplantation development
J Fragmentation
J Cryodamage
J Biopsy for PGD/PGS
14
Tours, April 11, 2008
Potency in invertebrates
Tunicata
Fate
Irreversible
15
Sea urchin
Plasticity
Reversible
Tours, April 11, 2008
5
Edwards
Edwards Mol Hum Reprod 1997,3:863-905
Edwards RBMOnline 2005,11:206-218
16
Tours, April 11, 2008
Where is the totipotent cell?
Splitting experiments cleavage stage embryos
z
z
z
z
Mouse 2-cell stage blastomeres
Some mouse 4-cell stage blastomeres in chimeras
Some rabbit and cow 4- and 8-cell stage blastomeres
Human: legal and ethical objections
17
Tours, April 11, 2008
Where is the totipotent cell?
J Splitting experiment human 4-cell stage embryo
J Approval Ethical Commity UZ Brussel May 11, 2006
J Informed consent
z > 8 mature oocytes: 1 for research
z Fresh mature oocytes if no sperm found in TESE
J ICSI with donor sperm
J Day 2: biopsy of 4 cells
J Each cell is manipulated and put into empty ZP
J In vitro culture day 2-day 6
18
Tours, April 11, 2008
6
Where is the totipotent cell?
z
Can each cell of the 4-cell stage human embryo develop into
blastocyst with ICM and TE?
z
Do the blastomere-derived blastocysts express ‘stemness
marker’ NANOG?
z
Can we derive pluripotent embryonic stem cells from a 4-cell
stage human embryo?
Klimanskaya Nature 2006,444:512
Blastomeres from human 8 cell-stage embryos: 2% derivation rate
19
Tours, April 11, 2008
Where is the totipotent cell?
Human 4-cell stage preimplantation embryo
20
Tours, April 11, 2008
Where is the totipotent cell?
In vitro development day 3-day 5
21
Tours, April 11, 2008
7
Where is the totipotent cell?
In vitro development day 6
22
Tours, April 11, 2008
Where is the totipotent cell?
NANOG expression in ICM cells
23
Tours, April 11, 2008
Where is the totipotent cell?
In vitro development day 2-day 6
Embryo
Day 2
Survived
Day 3
2-cell
Day 4
Compaction
Day 5
Cavity
Day 6
Full/expanded
blastocyst
(ICM/NANOG)
1
2
3
4
5
6
4
4
3
4
4
4
4
4
3
4
3
1
3
3
3
4
3
3
3
3
3
4
3
3
2 (1/ND)
3 (2/2)
3 (1/1)
4 (4/4)
3 (3/2)
1 (1/0)
Total
23
19
19
19
16 (12/9)
ND: not done because lost during fixation
4 cells develop into blastocysts with ICM and TE
no allocation
24
Tours, April 11, 2008
8
Where is the murine totipotent cell?
Prepatterning believers and non-believers
Believers
oocyte, zygote, 2-cell stage
Non-believers
inside-outside hypothesis
Tarkowsky 1967 J Em Exp Morph 18: 155-180
Piotrowska 2001, Development 128: 3739-3748: dye
Kurotaki 2007 Science 316: 719-215: photoconversion
in vitro and in vivo
Torres-Padilla 2006 Nature 445: 214-218
Gardner 1997 Development 124: 289-301
Gardner 2001 Development 128: 839-847
Gardner 2005 Hum. Reprod.
Piotrowska 2001 Nature 409: 517-521
Hiiragi 2006 Int. J. Dev. Biol. 50: 581-588
Motosugi 2006 PLoS 4: 799-804
Van de Velde Hum Reprod in press
25
Tours, April 11, 2008
Where is the pluripotent cell? hESC derivation from single
blastomeres of 4-cell stage embryos
J
J
J
J
J
Splitting experiment human 4-cell stage embryo
Day 2: biopsy of 4 cells
Each cell is manipulated and not put into empty ZP
In vitro culture day 4 (small morula)
Mouse feeder layer
26
Tours, April 11, 2008
Where is the pluripotent cell? hESC derivation from single
blastomeres of 4-cell stage embryos
Geens Mieke
Mateizel Ileana
Spits Claudia
Derivation 2/16 cells from 4 embryos:
VUB_26 QUATRO
passage 45
expresses stemness markers
teratoma, characterized (3 germ layers)
CGH: 46 XX, dup 7q33-ter, del 18q23-ter
VUB_27 PATRU
passage 19
expresses stemness markers
no teratoma yet
CGH: 46 XY
27
Tours, April 11, 2008
9
Where is the totipotent cell?
Human 8-cell stage preimplantation embryo
4 embryos
32 cells
14/32 little blastocysts
4/14 small ICM
28
Tours, April 11, 2008
Learning objectives
z
The cells of the 4-cell stage human embryo are
J potentially totipotent
J equivalent: individually able to develop into
blastocysts with ICM and TE
J no allocation to ICM or TE: plasticity
z
At least one cell of a 4-cell stage human
embryo can develop into ESC
J potentially pluripotent
29
Tours, April 11, 2008
Learning objectives
30
z
Definitions
J Totipotency
J Plasticity
z
Cell loss during preimplantation development
J Fragmentation
J Cryodamage
J Biopsy for PGD/PGS
Tours, April 11, 2008
10
Implantation rate after SET
z
z
Van Royen 1999 Hum Rep 14,2345-2349
Van Royen 2001 Hum Rep 16,326-332
J SET: IR 100% versus 0%
J Top quality on day 3: theoretical IR 47%
z 4 or 5 blastomeres on day 2
z at least 7 cells on day 3
z ≤ 20% fragmentation
z no multinucleation
Gerris 2002 Hum Rep 17,2626-2631
J Top quality on day 3: IR 35.1%
31
Tours, April 11, 2008
Fragmentation degree and pattern
Fragmentation day 3
Bl rate
SET IR
no
27.5%
48%
Fragment removal
0-5% minimal
19.9%
25%
38%
6-15% localized, small
1 blastomere
13.9%
29%
33%
6-15% scattered, small
8.8%
11%
31%
15-25% whole blastomeres
0%
0%
18%
>35% necrotic
0%
0%
Alikani 1999
Fertil Steril 71: 836-842
0
Stone Am J Obst Gynecol 2005,192:2014-2020
32
Tours, April 11, 2008
Fragmentation: degree and pattern
33
z
Alikani Fertil Steril 1999,71:836-842
J Large localized fragments:
loss of cells is correlated with poor implantation rate
J Fragment removal improves implantation rate
J No prospective randomized controlled study
z
Hardarson RBMOnline 2002,5:36-38
J Small scattered fragments are generated during divisions
J Fragments can be reabsorbed
Tours, April 11, 2008
11
UZ Brussel
Day 3 implantation rate (IR) according to cell stage
IR=FHB/ET
Overall 23.7%
8-cell stage 26.0%
40
35
4
30
5
6
25
7
8
20
12,16
15
9,10,11,13,14
10
Morula
all
5
n=1969
n=95
n=169
n=276
n=1019
n=172
n=64
n=126
0
n=53
Implantation rate (%)
2004-2007
1969 cycles
SET
Cell stage
34
Tours, April 11, 2008
UZ Brussel
Size of the blastomeres
normal
abnormal
35
Tours, April 11, 2008
UZ Brussel
Day 3 IR according to cell stage, fragmentation
and size of the blastomeres
8 cell-stage
Fragmentation
Size of the blastomeres: normal or abnormal
Implantation rate (%)
40
35
30
25
normal
20
abnormal
15
10
5
0
36
n=403
n=373
F0
F1
n=191
n=52
F2
F3
Tours, April 11, 2008
12
UZ Brussel
Fragmentation and size of the blastomeres
8 cells
F0
Irregular size blastomeres
8 cells
F2, scattered fragmentation
Regular size blastomeres
6 cells
F2, localized fragmentation
Regular size blastomeres
Loss of cytoplasm
(smaller cells)
Scattered fragments
Loss of cells
(cytoplasm+nucleus)
Localized fragments
37
Tours, April 11, 2008
Cell loss and fragmentation
1
0%
F0
2
12.5%
F1
3
25%
F2
4
37.5%
F3
5
50%
F4
6
62.5%
7
75%
8
87.5%
Loss of cells (cytoplasm+nuclei)
Localized fragments
6F2 ≤ 7F1 ≤ 8F0
6F3 ≤ 7F2 ≤ 8F1
6F4 ≤ 7F3 ≤ 8F2
7F4 ≤ 8F3
38
Tours, April 11, 2008
UZ Brussel: Day 3 implantation rate (%) according to
cell stage, fragmentation and size of the blastomeres
normal size
abnormal size
6 cells
7 cells
8 cells
100
100
100
90
90
90
80
80
80
70
70
70
6-cell stage (n=126)
60
50
40
30
20
10
0
(15 7
F0
15 15
F1
13 31
F2
7-cell stage (n=172)
60
50
4 26)
F3
8-cell stage (n=1019)
60
50
40
40
30
30
20
20
10
10
0
0
(20 11
F0
33 26
F1
22 30
F2
1
29)
F3
(344 59
F0
307 66
F1
107 84
F2
21 31)
F3
6F2 ≤ 7F1 ≤ 8F0
6F3 ≤ 7F2 ≤ 8F1
6F4 ≤ 7F3 ≤ 8F2
7F4 ≤ 8F3
39
Tours, April 11, 2008
13
UZ Brussel
Blastocyst development and fragmentation
Della Ragione, Verheyen
Rep Biol Endocrinol 2007,5:2
stage
IR
Top Bl3AA and Bl3AB
Top Bl4AA and Bl4AB
28.3%
52.4%
Fragmentation on day 3
P=0.03
100%
90%
80%
70%
60%
not-implanted
50%
implanted
40%
30%
20%
10%
0%
F0
and F1
≤10%
F0-F1
F2 and F3
>10-50%
F2-F3
40
Tours, April 11, 2008
Learning objectives
z
Definitions
J Totipotency
J Plasticity
z
Cell loss during preimplantation development
J Fragmentation
J Cryodamage
J Biopsy for PGD/PGS
41
Tours, April 11, 2008
Damage after cryopreservation
42
z
Veiga Hum Reprod 1987,2:321-323
J Case report 1/4, pregnancy
z
Van den Abbeel Hum Reprod 1997,12:2006-2010
J Case report 3/4, born
J Case report 1/4, biochemical pregnancy
J obstructive (and/or toxic) effect damaged cell
z
Van Landuyt and Van den Abbeel, in preparation
J Case report 4/8, born
J Case report 4/8, clinical pregnancy
Tours, April 11, 2008
14
Damage after cryopreservation
Edgar 2007 RBMOnline 14: 718-723
Tang 2006 Hum reprod 21: 1179-1183
ET Day 2
IR sFET
ET Day 3
IR sFET
4/4
3/4
2/4
26.0%
27.5%
9.4%
8/8, 7/7, 6/6
7/8, 6/7, 5/6
6/8, 5/7, 4/6
10.3%
11.1%
4.2%
Resumption of mitosis
43
Tours, April 11, 2008
Damage after cryopreservation
Implantation rate (%)
Van Landuyt and Van den Abbeel
2004-2007: 530 sFET cycles,
cryo day 3, ET day 4
25
20
15
0
-1
10
-2
5
0
8-cell stage
n=175 n=43
n=17
overall
n=382 n=86
n=34
Resumption of mitosis
44
Tours, April 11, 2008
Damage after cryopreservation
Removal of lysed blastomeres on day 3
no prospective randomized study
J Nagy Fertil Steril 2005,84:1606-1612
J Rienzi Fertil Steril 2005,84:888-894
Cleavage rate
IR
LBR
45
0 lost
1-2 lost
3-4 lost
P value
81.3%
21.6%
34.4%
76.8%
21.4%
34.0%
67.5%
17.2%
29.4%
NS
NS
NS
Tours, April 11, 2008
15
Learning objectives
z
Definitions
J Totipotency
J Plasticity
z
Cell loss during preimplantation development
J Fragmentation
J Cryodamage
J Biopsy for PGD/PGS
46
Tours, April 11, 2008
Biopsy for PGD/PGS
Hardy 1990 Hum Reprod 5,708-714
ICM/TE ratio is not altered
Goossens, De Rycke and De Vos 2007
Hum Reprod in press
Prospective randomized controlled study
n=592
1-cell versus 2-cell biopsy
Biopsy day 3, ET day 5
Life birth rate
no diagnosis, misdiagnosis
47
Tours, April 11, 2008
Biopsy for PGD/PGS
Randomized controlled study
Goossens, De Rycke and De Vos
Prospective randomized controlled study, 1-cell versus 2-cell biopsy
Cycles
Transfer rate
Implantation rate
Miscarriage rate
LBR
1 cell
2 cells
P value
288
77.1%
23.5%
34.0%
26.2%
304
75.3%
17.3%
28.0%
22.8%
NS
NS
NS
NS
1 less delivery following 2-cell biopsy every 33 cycles
In vitro development (Bl rate):
1-cell biopsy > 2-cell biopsy (p=0.007)
Embryo quality on day 3 (p<0.0001)
48
Tours, April 11, 2008
16
Biopsy for PGD
Sub-analysis SET
De Vos Anick
ICSI, PGD/PGS, 1-cell versus 2-cell biopsy
LBR rate (%)
35
1/4
30
25
20
1/10
15
-1
-2
eSET
= cryo ≥1
10
5
0
8-cell stage
overall
n=46 n=58
n=90 n=106
Case report 8-4: born
49
Tours, April 11, 2008
Conclusions
z
The cells of the 4-cell stage human embryo are
J Potentially totipotent
J Equivalent (plasticity)
z
Severe cell loss (>25%) is detrimental for implantation
J Fragmentation: scattered versus localized
J Cryodamage: 8/8 = 7/8 ≥ 6/8
J Biopsy for PGD/PGS: 8-1 ≥ 8-2
50
Tours, April 11, 2008
Cell loss during preimplantation development
z
51
Two hypothesis on cell loss:
J Loss of large volume (nuclei and/or cytoplasm)
J Abnormal cell-cell contact (obstruction and/or toxic):
compaction
cavitation
blastulation
Tours, April 11, 2008
17
Cell loss during preimplantation development
z
Clinical experiments
J Look carefully and score
J Time lapse
J Randomized controlled studies removing
z fragments
z cryodamaged cells
J Combinations (fragmentation and biospy,
fragmentation and cryodamage)
52
Tours, April 11, 2008
Thanks
Geertrui Bocken
Heidi Van Ranst
Nadine Franceus
An Vankelecom
Sabrina Vitrier
Griet Meersdom
Saar Bijnens
Johan Sterckx
Greet Cauffman
Mieke Geens
Ileana Mateizel
Claudia Spits
An Verloes
Walter Meul
Yves Guns
Martine De Rycke
Karen Sermon
Josiane Van der Elst
Etienne Van den Abbeel
Anick De Vos
Lisbet Van Landuyt
Greta Verheyen
Ronny Janssens
Herman Tournaye
Paul Devroey
Inge Liebaers
53
Tours, April 11, 2008
Stemness markers in hESC lines
Richards 2004 STEM CELLS 22: 51-64
+ KLF4 + c-MYC
Takahashi 2007 Cell 131:1-12
Yu 2007 Science
= reprogramming differentiated
somatic cells into pluripotent state
54
Tours, April 11, 2008
18
Proof of the model?
Monozygotic bichorionic biamniotic twinning
55
Tours, April 11, 2008
Monozygotic bichorionic biamniotic twinning
and ART
Massip et al. 1983
Abnormal ZP thinning
Alikani Hum Reprod 2003,18:1937-1943
Hall Lancet 2003,362:735-743
Allegra J Ass Reprod Genet 2005,22:437-441
56
Tours, April 11, 2008
19