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Derivation of ES cell lines in non-murine mammalian species
Marielle Afanassieff
To cite this version:
Marielle Afanassieff. Derivation of ES cell lines in non-murine mammalian species. Visite d’un labo- ratoire de l’académie chinoise des sciences, Jul 2007, Beijing, China. �hal-02816721�
Stem Cell and Brain Research Institute Inserm U846
Marielle AFANASSIEFF
LYON PARIS
Transversal project of Stem Cell and Brain Research Institute
Dopaminergic
neurons Grafts
Model of Parkinson disease in monkey
(MPTP monkeys)
Recovery after graft
Behavioral and
electrophysiological studies in behaving monkeys
Anatomical brain imaging techniques Derivation of
ESC lines
Strategies for coaxing neuronal differentiation
HPLC Quantification of enzymes activities…
In vivo injection into central structures,
axonal tracing…
Embryonic Stem Cells
Mechanisms underlying proliferation and
pluripotency
Transversal project of Stem Cell and Brain Research Institute
Dopaminergic
neurons Grafts
Model of Parkinson disease in monkey
(MPTP monkeys)
Recovery after graft
Behavioral and
electrophysiological studies in behaving monkeys
Anatomical brain imaging techniques Derivation of
ESC lines
Strategies for coaxing neuronal differentiation
HPLC Quantification of enzymes activities…
In vivo injection into central structures,
axonal tracing…
Embryonic Stem Cells
Mechanisms underlying proliferation and
pluripotency
PrimaStem
PrimaStem
Research-based biotechnology platform
Derivation of ESC lines in non murine mammalian species
PrimaStem
Research-based biotechnology platform
Derivation of ESC lines in non murine mammalian species
ü Embryonic Stem Cells (ESC)
ü Derivation of rhesus Lyon-ES line
ü Isolation and characterization of rabbit and goat
ES-like cells with human ESC features
Embryonic Stem Cells (ESC)
Proprieties of ESC
Self-Renewal Differentiation
Blastocyst ESC
Endoderm Mesoderm Ectoderm
Proprieties of ESC
Self-Renewal Differentiation
Blastocyst ESC
Endoderm Mesoderm Ectoderm
ESC ESC
In vitro
genetic modifications
Injection into donor blastocysts
Chimaeric and transgenic mice
ü Morphology : compact cells, high nucleus/cytoplasm ratio
Characteristics of ESC
Mouse ESC
Evans and Kaufman 1981
Chicken ESC
Pain et al. 1996
Human ESC
Thomson et al. 1995
Monkey ESC
Thomson et al. 1996
Rabbit ESC
Wang et al. 2006
ü Morphology : compact cells, high nucleus/cytoplasm ratio ü Enzymatic activities : Alkaline Phosphatase, Telomerase ü Cell surface markers :
Mouse : SSEA-1
Primates : SSEA-3, SSEA-4, TRA-1-60, TRA-1-81
Rabbit : SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 ü Nuclear markers : Oct4, Nanog, Sox2
ü Growth rate : doubling time from 10 to 24 hours ü Karyotype : normal diploid
Characteristics of ESC
Mouse ESC
Evans and Kaufman 1981
Chicken ESC
Pain et al. 1996
Human ESC
Thomson et al. 1995
Monkey ESC
Thomson et al. 1996
Rabbit ESC
Wang et al. 2006
Interests of ESC
ü Powerful tool for transgenesis
Genetic engineering
Addition or deletion of genes Defined mutation
Unlimited source of nuclei for cloning by nuclear transfer
Interests of ESC
ü Powerful tool for transgenesis
Genetic engineering
Addition or deletion of genes Defined mutation
Unlimited source of nuclei for cloning by nuclear transfer
ü Useful tool for fundamental studies
Differentiation pathways Embryonic development Gene functions
Interests of ESC
ü Powerful tool for transgenesis
Genetic engineering
Addition or deletion of genes Defined mutation
Unlimited source of nuclei for cloning by nuclear transfer
ü Useful tool for fundamental studies
Differentiation pathways Embryonic development Gene functions
ü Helpful tool for biotechnology
Animal models of human diseases Animal bioreactors
Interests of ESC
ü Powerful tool for transgenesis
Genetic engineering
Addition or deletion of genes Defined mutation
Unlimited source of nuclei for cloning by nuclear transfer
ü Useful tool for fundamental studies
Differentiation pathways Embryonic development Gene functions
ü Helpful tool for biotechnology
Animal models of human diseases Animal bioreactors
ü Promised tool for therapy in human
Cell replacement therapy of degenerative diseases Gene therapy of genetic diseases
Collaboration between LIF and BMP signaling to maintain mouse ESC pluripotency
Sun et al. 2006
Collaboration between FGF and Activin/Nodal signaling to maintain primate ESC pluripotency
Sun et al. 2006
Critical role of Wnt signaling in maintaining both mouse and primate ESC pluripotency
Sun et al. 2006
Relationship between GSK3 β , Myc and p53 in maintenance of ESC self-renewal
Sun et al. 2006
Regulatory core controlling of ESC pluripotency
Sun et al. 2006
==> Regulatory core maintains self-renewal of ESC by activation of ESC-specific genes, repression of cell type-specific genes, and
regulation of cell cycle and cell death.
Conclusion
==> Self-renewal and pluripotency of ESC relies on a hierarchical
regulatory structure including :
Conclusion
==> Self-renewal and pluripotency of ESC relies on a hierarchical regulatory structure including :
ü signal transduction pathways to transfer multiple extracellular signals into the cell;
Conclusion
==> Self-renewal and pluripotency of ESC relies on a hierarchical regulatory structure including :
ü signal transduction pathways to transfer multiple extracellular signals into the cell;
ü intrinsic regulatory factors forming the regulatory core, which integrates extracellular signals received and controls the expression of dowstream genes;
Conclusion
==> Self-renewal and pluripotency of ESC relies on a hierarchical regulatory structure including :
ü signal transduction pathways to transfer multiple extracellular signals into the cell;
ü intrinsic regulatory factors forming the regulatory core, which integrates extracellular signals received and controls the expression of dowstream genes;
ü target genes regulated by the core regulatory module, including both genes initiating differentiation and factors involved in long-term self-renewal.
Conclusion
==> Self-renewal and pluripotency of ESC relies on a hierarchical regulatory structure including :
ü signal transduction pathways to transfer multiple extracellular signals into the cell;
ü intrinsic regulatory factors forming the regulatory core, which integrates extracellular signals received and controls the expression of dowstream genes;
ü target genes regulated by the core regulatory module, including both genes initiating differentiation and factors involved in long-term self-renewal.
==> Isolation of ESC lines relies on the adaptation of epiblast stem
cells to in vitro conditions maintaining Oct-4, Nanog and
Sox2 gene expression and supporting self-renewal
Derivation of rhesus Lyon-ES line
Derivation of primate ESC lines
In vitro fertilization by ICSI : IntraCytoplasmic Sperm Injection
Spermatozoa
Fertilization (55%)
Embryo culture
Blastocyst (15 to 30%)
N=34
Monkey embryo production and culture
Metaphase I Metaphase II FSH (7-9 days) rh hCG
Menses
After 27-32h collection of mature oocytes (41%)
Superovulation
Passage 6
Inner Cell Mass cell culture
Blastocyst Immunosurgery ICM on MEF
Passage 2 Passage 6
Lyon-ES cells Passage 1
ICM derived cells ICM
Morphology of Lyon-ES Cell
Monkey ORMES
Monkey Lyon-ES
Human
H1
Enzymatic activities in Lyon-ES Cells
Alkaline phosphatase
activity
Telomerase activity
ORMES Lyon-ES
Anti-SSEA-4
Anti-TRA-1-60
Expression of primate ESC markers by Lyon-ES Cells
Expression of pluripotent markers by Lyon-ES Cells
Anti-Oct4 Anti-Nanog
RT-PCR:
O = ORMES RNA L = Lyon-ES RNA
O L O O L O L O L
O L O L O L O L O L
C-
Normal et stable karyotype of Lyon-ES Cells
Chromosomal alterations in long term cultures of primate ES cells:
- affect the growth rate or the capacity to differentiate;
- cause abberant gene expression.
Difficult and laborious manual passaging
Carreful monitoring of the karyotype
Cell cycle of Lyon-ES cells
Lyon-ES cells Differentiated cells
Differentiation G1
G2 S
M ==> Lyon-ES cells show
typical ESC cell cycle G1
G2 S M
Number of cells
Quantity of DNA
Number of cells
Quantity of DNA
In vitro study of Lyon-ES cell pluripotency
Embryoid Bodies
D7
D21
D28 Mesoderm
Ectoderm
Endoderm ESC
1: ESC 2: EB D2 3: EB D7 4: EB D21 5: EB D28
In vivo study of Lyon-ES cell pluripotency
Mesoderm Ectoderm
Endoderm
A: GFAP B: Nestin C: Rosette
D: Glucagon E: GATA4 F: HNF3β
G: Desmin
H: Alizarine Red I: Oil Red
Injection of Lyon-ES cells in testis of SCID mice
==> Tumor development 6 weeks later
Lyon-ES cell labeling using Tau-GFP expressing lentiviral vector
Manual enrichment Cell sorting + cloning
4 days post-infection
10% GFP+ ESC 85% GFP+ ESC 100% GFP+ ESC
Tau-GFP expression after induction of differentiation into neural lineage
Rosette Glial differentiation Neuronal differentiation
Conclusion
ü Lyon-ES cell line shows all the characteristics of a primate ES cell line :
- Morphology
- Cell surface markers - Molecular markers - Enzymatic activities - Stable Karyotype - Immortality
- Cell cycle
- Pluripotency in vitro and in vivo
Conclusion
ü Lyon-ES cell line shows all the characteristics of a primate ES cell line :
- Morphology
- Cell surface markers - Molecular markers - Enzymatic activities - Stable Karyotype - Immortality
- Cell cycle
- Pluripotency in vitro and in vivo
ü Lyon-ES cell line stably expressing GFP gene is an useful tool for :
- Monitoring induction of in vitro differentiation - Monitoring cell grafts in animals
- Monitoring creation of chimera after ESC injection in blastocysts
Isolation and characterization of rabbit and goat ES-like cells
with human ESC features
Rabbit and goat ESC derivation
Blastocyst Blastocyst without
mucus coat
Blastocyst without zona pellucidae
Inner Cell Mass
Culture Medium :
KO-DMEM + 10% FBS + 10% KO-SR + FGF2 (medium using to derive monkey Lyon-ES line)
Pronas e
Mechanic elimination al
Immunosurger y
Culture on Feeder cells
Rabbit morula Culture
Goat blastocyst Morula
Rabbit blastocyst without zona pellicidae
Rabbit ICM Rabbit ICM after 20h culture on feeder cells
Morphology of primary and secondary colonies
Primary culture of rabbit ICM:
appearance of outgrowths after 5 days Rabbit ES-like P2 colony Mechanical
dissociation
Collagenase and Mechanical dissociation
Goat ES-like P2 colony Primary culture of goat ICM:
appearance of outgrowths after 3 days
Morphology of secondary ES-like colonies
x10
Goat ES-like colony
x20
Rabbit ES-like colony
x10
x20
Human ES colony
x10
x20
Ø Flat colonies of compact cells
Ø High nucleus/cytoplasm ratio and proeminent nucleoli
Alkaline phosphatase activity in ES-like cells
AP Test
Rabbit ES-like colony
Goat ES-like colony
AP Test
Rabbit
Mouse Goat
Oct4 expression in blastocyst cells
Ø High Oct4 expression in ICM cells
Ø Lower Oct4 expression in trophectoderm cells
Nucleus labelling by propidium
iodide
Anti-Oct4 immuno- flurescence
confocal microscopy
X40
X40
X40 X40
X25 X25
Oct4 expression in rabbit ES-like cells
Oct4
x20 x20 x10
x10
x10
x10
Ø Loss of Oct4 expression with ES-like cell differentiation
Ø Self-renewal of ES-like cells is not sustained in applied culture conditions
x10
Contrast of Phase Hoechst Anti-Oct4
x10
Passage 1 Passage 2
X10
Passage 4
X10
Passage 6
x10
x10 x10
X10
X10 X20
X20
X10 X10
X10
Oct4 expression in goat ES-like cells
Passage 1 Passage 2
Contrast of Phase
Hoechst
Anti-Oct4
X20 X10
X10
X10 X20
X20
X20
X20 X20
Type of embryos
Number of blastocysts
Number of isolated
ICM
Number of plated
ICM
Number of P1 outgrowths
Number of P2 colonies
Number of passages
Frozen rabbit blastocysts
644 336
52%
216 64%
54/177 30%
21/47
45% P3
Fresh rabbit blastocysts
461 326
71%
280 86%
156/265 59%
107/154
69% P9
Total rabbit blastocysts
1105 662
60%
496 75%
210/442 47%
128/201 64%
P3 54%
P8 5%
Frozen goat blastocysts
78 46
59%
40 87%
11/25 44%
1/11
9% P2
Fresh goat blastocysts
306 233
76%
161 69%
61/155 39%
16/57
28% P3
Total goat blastocysts
384 279
73%
201 72%
72/180 40%
17/68
25% P3 24%
Efficiency of isolation of ES-like Cells
Ø Isolation of rabbit and goat ES-like cells with human ESC features.
Ø Very low efficiency of ES-like cell isolation.
Ø Spontaneous differentiation of the ES-like cells after three or eight passages according to the specie.
Ø A phenomenon associated with the loss of Oct4 expression.
Conclusion
Ø Isolation of rabbit and goat ES-like cells with human ESC features.
Ø Very low efficiency of ES-like cell isolation.
Ø Spontaneous differentiation of the ES-like cells after three or eight passages according to the specie.
Ø A phenomenon associated with the loss of Oct4 expression.
==> Development of two different strategies to increase self-renewal of our ES-like cells:
1. Improvement of derivation and culture conditions 2. overexpression of transcription factors involved in sustaining pluripotency in mouse and human ESC
Conclusion
Improvement of derivation and culture conditions
Comparison of two media :
French ES medium : KO-DMEM + 10% FBS + 10% KO-SR + 8 ng/ml FGF2 Chinese F12 medium: DMEM/F12 + 20% SR + 8 ng/ml FGF2
Results :
Immunosurgery of 347 blastocysts ==> culture of 270 ICM (78%)
F12 medium gives more and nicer P1 outgrowths and more P2 ES-like colonies ES medium allows to obtain less ES-like colonies but nicer and until passage 4
Conclusion :
F12 medium does not sustain self-renewal of our ES-like cells
Chinese ES cell Medium
Medium F12 medium ES medium
Plated ICM 133 123
P1 with
outgrowths 91 (68%) 64 (52%)
P2 with ES-like
colonies 31 (34%) 30 (47%)
P3 with ES-like
colonies 0 (0%) 6 (20%)
Mouse ESC LIF/Stat3
Primate ESC
BMP4/Smad FGF Activin/Nodal bcatenin
LIF Serum FGF2
8ng/ml
Activin 10 ng/ml Nodal 1 ng/ml
BIO 2 µ M
Effect of Activin/Nodal and BIO on ES-like cell culture
Mouse ESC LIF/Stat3
Primate ESC
BMP4/Smad FGF Activin/Nodal bcatenin
LIF Serum FGF2
8ng/ml
Activin 10 ng/ml Nodal 1 ng/ml
BIO 2 µ M Comparison of Chinese F12 and French ES media +/- factors
Results :
Immunosurgery of 141 blastocysts
==> culture of 102 ICM
Conclusion :
Addition of factors (Activin, Nodal and BIO) is not sufficient to maintain our
ES-like cells in culture.
Effect of Activin/Nodal and BIO on ES-like cell culture
Medium ES ES +
Factors F12 F12 + Factors Plated ICM 21/21 28/30 16/21 30/30
P1 with outgrowths
12 (57%)
28 (46%)
10 (62%)
15 (50%) P2 with ES-like
colonies
10 (83%)
12 (92%)
4 (40%)
9 (60%) P3 with ES-like
colonies
6 (60%)
1 (8%)
3 (75%)
0 (0%)
Culture of embryos
1-cell embryos Adhesion of mucus cells 8-cell embryos
Results:
Culture of 132 1-cell embryos ==> 111 blastocysts (84%)
High development rate of blastocysts in culture
Blastocysts
Isolation of ICM by trypsin
Results:
84 blastocysts from 1-cell embryo culture ==> 59 ICM after Trypsin (70%) 78 blastocysts from thawed morula ==> 42 ICM after Trypsin (54%)
Trypsin versus immunosurgery :
ICM are less visible and could be easily damaged by trypsin ==> Test ICM isolation by dispase
Blastocysts after trypsin Blastocysts after mechanical dissociation ICM on feeder cells
ü Comparison of 129 MEF and CF1 MEF:
Immunosurgery of 103 blastocysts ==> culture of 60 ICM
Type and concentration of feeder cells
Type of
feeder Plated ICM P1 outgrowths P2 with ES- like colonies 129 MEF 29 (100%) 15 (51%) 11 (37%) CF1 MEF 31 (100%) 17 (55%) 8 (26%)
No difference between the two types of feeder cells
ü Test higher concentration of feeder cells:
4 times more : 1.5x105 cells/well (usually 4x104 cells/well, with 4-well plates) ==> appearance of colonies similar to rabbit ESC described by Shufen Wang
ü Comparison of 129 MEF and CF1 MEF:
Immunosurgery of 103 blastocysts ==> culture of 60 ICM
Type and concentration of feeder cells
Type of
feeder Plated ICM P1 outgrowths P2 with ES- like colonies 129 MEF 29 (100%) 15 (51%) 11 (37%) CF1 MEF 31 (100%) 17 (55%) 8 (26%)
No difference between the two types of feeder cells
Concentration of feeder cells seems to play
an important role in deriving rabbit ES cells
Nanog
Oct4
Sox2
Strategies of overexpression of pluripotency genes
Transcription factors involved in sustaining pluripotency in mouse and human ESC
Nanog
Oct4
Sox2
Strategies of overexpression of pluripotency genes
Transcription factors involved in sustaining pluripotency in mouse and human ESC
Ø SIV-derived lentiviral vectors
In collaboration with FL Cosset (Inserm U758, ENS Lyon, France) Test several amphotropic envelopes
Test different promoters
Nanog
Oct4
Sox2
Strategies of overexpression of pluripotency genes
Transcription factors involved in sustaining pluripotency in mouse and human ESC
Ø SIV-derived lentiviral vectors
In collaboration with FL Cosset (Inserm U758, ENS Lyon, France) Test several amphotropic envelopes
Test different promoters Ø Tat-mediated protein transduction
In collaboration with F Edenhofer (Bonn University, Germany)
Test TAT-Nanog protein
Viral RNA
Env Pol
Gag
Viral membrane
Lentiviral cycle
Ψ
SIV-derived lentiviral vectors
ΔΨ
CMV polyA
ΔΨ
CMV polyA
Ψ
CMV Promoter GFP
SIV
Vectors
Amphotropic envelopes VSV-G
Ha/Na RD/RT LCMV 4070A Promoters
E1a CAG
PGK
Lentiviral infection of rabbit blastocyst cells
ICM Passage 1 48h culture
Outgrowths Passage 1 5 day culture
ES-like colony Passage 2 48h culture
VSV-G pseudotyped vector expressing CAG-GFP transgene
Contrast of phase Immunofluorescence
Ø GFP expression in ES-like cells following lentiviral infection of blastocyst cells
Lentiviral infection of goat blastocyst cells
VSV-G pseudotyped vector expressing CAG-GFP or PGK-GFP transgene
Contrast of phase Immunofluorescence
CAG promoter PGK promoter
Outgrowths Passage 1 3 day culture
Outgrowths Passage 1 3 day culture
Outgrowths Passage 1 7 day culture
Ø Only PGK promoter induces GFP expression in goat ES-like cells
ü Technology which allows the entry of biologically active proteins into mammalian cells with high efficiency
ü Technology based on the membrane penetration propriety of the TAT protein of HIV
ü TAT-Oct4 and TAT-Nanog together sustain self-renewal and pluripotency of mouse ESC in the absence of LIF or feeder cells
ü Addition of the TAT-fusion proteins to culture medium ==> Absence of genetic modifications of the cells
TAT-mediated protein transduction
Myoblast transducted with FITC-labeled TAT peptide
Myoblast transducted with Rhodamine-labeled TAT peptide
Ø TAT-Nanog protein increases the ability of the cells to : - give new colonies
- maintain pluripotency in culture
P2 colony
in Basic Control Medium
P2 colony
in Basic Nanog Medium 0
2 4 6 8 10 12 14 16
P2 P3 P4 P2 P3 P4
Basic Témoin Basic Nanog SR Témoin SR Nanog
Number of Passages Number of
colonies
6-9 3-9 0-6 4-4 3-3 0-2 Number of samples
TAT-Nanog protein transduction of rabbit blastocyst cells
Basic Control Basic Nanog SR Control SR Nanog
Ø The quality and the density of feeder cells is crucial for the rabbit ESC derivation Ø The culture of embryos and the technique of isolation of ICM could be also
important
Conclusion
Ø The quality and the density of feeder cells is crucial for the rabbit ESC derivation Ø The culture of embryos and the technique of isolation of ICM could be also
important
==> Improvement of the derivation and culture conditions according to the technique of Shufen Wang
Conclusion
Ø The quality and the density of feeder cells is crucial for the rabbit ESC derivation Ø The culture of embryos and the technique of isolation of ICM could be also
important
==> Improvement of the derivation and culture conditions according to the technique of Shufen Wang
Ø Expression of the GFP reporter gene in rabbit or goat blastocyst cells following lentiviral infection of Inner Cell Mass
Ø Efficiency of used promoter is different according to the specie:
CAG promoter for rabbit cells and PGK promoter for goat cells
Conclusion
Ø The quality and the density of feeder cells is crucial for the rabbit ESC derivation Ø The culture of embryos and the technique of isolation of ICM could be also
important
==> Improvement of the derivation and culture conditions according to the technique of Shufen Wang
Ø Expression of the GFP reporter gene in rabbit or goat blastocyst cells following lentiviral infection of Inner Cell Mass
Ø Efficiency of used promoter is different according to the specie:
CAG promoter for rabbit cells and PGK promoter for goat cells ==> Use of lentiviral vector to overexpress Oct4, Nanog and Sox2 genes in rabbit ES-like cells
Conclusion
Ø The quality and the density of feeder cells is crucial for the rabbit ESC derivation Ø The culture of embryos and the technique of isolation of ICM could be also
important
==> Improvement of the derivation and culture conditions according to the technique of Shufen Wang
Ø Expression of the GFP reporter gene in rabbit or goat blastocyst cells following lentiviral infection of Inner Cell Mass
Ø Efficiency of used promoter is different according to the specie:
CAG promoter for rabbit cells and PGK promoter for goat cells ==> Use of lentiviral vector to overexpress Oct4, Nanog and Sox2 genes in rabbit ES-like cells
Ø TAT-Nanog protein increases the ability of rabbit ES-like cells to maintain pluripotency in culture
Conclusion
Ø The quality and the density of feeder cells is crucial for the rabbit ESC derivation Ø The culture of embryos and the technique of isolation of ICM could be also
important
==> Improvement of the derivation and culture conditions according to the technique of Shufen Wang
Ø Expression of the GFP reporter gene in rabbit or goat blastocyst cells following lentiviral infection of Inner Cell Mass
Ø Efficiency of used promoter is different according to the specie:
CAG promoter for rabbit cells and PGK promoter for goat cells ==> Use of lentiviral vector to overexpress Oct4, Nanog and Sox2 genes in rabbit ES-like cells
Ø TAT-Nanog protein increases the ability of rabbit ES-like cells to maintain pluripotency in culture
==> Improvement of the TAT-mediated protein transduction method
Conclusion
Pierre Savatier
Suzy Markossian
Florence Wianny
ISARA Lyon, France
Production of rabbit embryos Thierry Joly
Pascal Salvetti
INRA Nouzilly, France
Production of goat embryos Pascal Mermillot
Gérard Baril Nati Poulin
INSERM U758 ENS Lyon, France
Lentiviral vectors François-Loïc Cosset
Didier Nègre
Bonn University, Germany
TAT-fusion proteins Frank Edenhofer
Mickaël Peitz
Thanks to…
Guillaume Marcy Colette
Dehay
