PRE-CONGRESS COURSE 13

Of stem cells and gametes:

more similarities than
differences?
Special Interest Group Stem Cells
Munich - Germany, 29 June 2014

coverPCC13.indd 2 8/05/2014 11:36:45



Ofstemcellsandgametes:more
similaritiesthandifferences

Munich,Germany
29June2014

Organisedby
TheESHRESpecialInterestGroupStemCells




 Contents

Coursecoordinators,coursedescriptionandtargetaudience Page5

Programme Page7

Speakerscontributions

Beforethegamete,therewastheprimordialgermcell
PetraHajkovaUnitedKingdom Page9

Dazlin'GermCellsandPluripotentStemCells
NielsGeijsenU.S.A. Page23

CurrentstatusofinvitrodifferentiationofHPSCintofemalegametes
SusanaM.ChuvadeSousaLopesTheNetherlands Page33

InvitrodifferentiationofhPSCintomalegametes:currentstatusand
theroadahead
CristinaEguizabalSpain Page43

Functionalcharacterizationofadultovaryderivedoogonialstemcells
inmice,monkeysandwomen
JonathanL.TillyU.S.A. Page59

Domitoticallyactivefemalegermlineprogenitorsexistinpostnatal
mouseovaries?
KuiLiuSweden Page71

Spermatogoniastemcellsandfuturefertility
AnsvanPeltTheNetherlands Page84

Stemcellbasedapproachestorestorespermatogenesisinmonkeys
StefanSchlattGermany Page99

UpcomingESHRECampusCourses Page116

Notes Page117

Page 3 of 124
Page 4 of 124
Coursecoordinators

KarenSermon(Belgium),RitaVassena(Spain)

Coursedescription

Thisisanadvancedcourseonthelatestdevelopmentsinthedifferentiationofhumanpluripotent
stemcells,bothembryonic,adultandinduced,intogametes,bothoocytesandsperm.Thecourse
startswithacomparisonofdifferentshadesofstemcells,andwhattheirpotentialofdifferentiation
towardsgametesare.Thedifferentiationintoprimordialgermcellsstartingfromtothe
preimplantationembryoisdrawn.Twospeakerseachthendescribecurrentknowledgeon
differentiationofhPSCintoeithermaleorfemalegametes.Alternativeroutes,suchasfromtheadult
ovaryandtestesaredescribednext.

Targetaudience

Targetaudiencearemainlyscientistsworkinginthefieldofstemcells,butalsoclinicalembryologists
withageneralinterestinfundamentalembryologyandcliniciansinterestedinalternativewaysto
obtaindonorgametes.

Page 5 of 124
Page 6 of 124
 Scientificprogramme

Chairmen:KarenSermonBelgiumandCristinaEguizabalSpain

09:0009:30 Beforethegamete,therewastheprimordialgermcell
PetraHajkovaUnitedKingdom
09:3009:45 Discussion
09:4510:15 Dazlin'GermCellsandPluripotentStemCells
NielsGeijsenU.S.A.
10:1510:30 Discussion

10:3011:00 Coffeebreak

Chairmen:AnnaVeigaSpainandBjornHeindryckxBelgium

11:0011:30 CurrentstatusofinvitrodifferentiationofHPSCintofemalegametes
SusanaM.ChuvadeSousaLopesTheNetherlands
11:3011:45 Discussion
11:4512:15 InvitrodifferentiationofhPSCintomalegametes:currentstatusandtheroad
ahead
CristinaEguizabalSpain
12:1512:30 Discussion

12:3013:30 Lunch

Chairmen:KarenSermonBelgiumandRitaVassenaSpain

13:3014:00 Functionalcharacterizationofadultovaryderivedoogonialstemcellsinmice,
monkeysandwomen
JonathanL.TillyU.S.A.
14:0014:15 Discussion
14:1514:45 Domitoticallyactivefemalegermlineprogenitorsexistinpostnatalmouseovaries?
KuiLiuSweden
14:4515:00 Discussion

15:0015:30 Coffeebreak

Chairmen:RitaVassenaSpainandFilippoZambelliItaly

15:3016:00 Spermatogoniastemcellsandfuturefertility
AnsvanPeltTheNetherlands
16:0016:15 Discussion
16:1516:45 Stemcellbasedapproachestorestorespermatogenesisinmonkeys
StefanSchlattGermany
16:4517:00 Discussion

Page 7 of 124
Page 8 of 124
 Before the gamete, there was the primordial germ cell

Petra Hajkova
Reprogramming and Chromatin Group
MRC Clinical Sciences Centre
London UK

Conflictofinterest:

Presenterdeclaresnoconflictofinterest.

Learningobjectives

Keyaspectsofgermline developmentandepigeneticproperties

Conceptandmechanisticoutlineofgermline epigeneticreprogramming

Derivationandpropertiesofembryonicgermcells(EGcells)

Relationshipbetweengermline,pluripotency andstemcells

Page 9 of 124
 Mousegermlinedevelopment

HiroyukiSasaki&YasuhisaMatsui
NatureReviewsGenetics9,129140(February2008)

Specificepigeneticpropertiesofgermline

Mousedevelopment&genomicimprinting

SuraniandBarton,1983,Barton,Surani,Norris1984, Hajkovaetal,2002;
McGrathandSolter,1984 Leeetal,2002

Page 10 of 124
 Early mouse development and
appearance of primordial germ cells (PGCs)

E6.0 6.25 7.5 8.0 8.5 9.5 E12.5

stella
Blimp1 sox2
Prdm14 nanos3 nanog reprogramming

Tcfap2c esg1

epiblast fragilis
migration into the Loss of
specification genital ridge Blimp1

Epigeneticprogrammingofgermlineoccursin2distinctsteps

~ 3500PGCs
~ 45PGCs

E6.0 6.25 7.5 8.0 8.5 9.5 E12.5

epiblast specification migration reprogramming

H3K9me2 of
Establishment H3K27me3
distinct (imprint erasure, X-reactivation,
chromatin signature DNA demethylation)
H2A/H4 R3me2s

PGCs undergo genome

wide DNA
demethylation in
genital ridges

DNA methylation
analysis : Bisulphite
sequencing

E 10.5 E 11.5 E 12.5 E 13.5 Hajkova et al, 2002

Page 11 of 124
 BisseqorLC/MSdataongermlinedemethylation

Impact on nuclear architecture

10.5 11.5 12.5 dpc

ChromatinchangesingonadalPGCs overview

Hajkovaetal,2008

Page 12 of 124
 ?
Chromatin DNA
remodelling demethylation

DNAdemethylation Chromatinremodelling

LossofH1
ssDNAbreaks
LossofH2A.Z
(chromatinboundXRCC1)
PARpolymers Lossofmanyhistone
ActivationofBER modifications
Translocationofhistone
chaperones

DNAdemethylation(DNArepair)

Erasureofhistonemodifications
(histonereplacement)
Hajkovaetal,2008

MolecularmechanismsofepigeneticreprogramminginPGCs?

Manyquestionsremaining..

PassivelossofDNAmethylation
(replicationwithoutmaintenace oftheDNAmethylationpattern)?

ActiveDNAdemethylation (Baseexcisionrepairpathway)?

Conversionof5mC(5methylcytosine)to5hmC(5hydroxymethylcytosine)
drivenbyTet1enzyme?

Tet1

Page 13 of 124
 Germlineandpluripotency

Germcellsandpluripotency

Primordialgermcells(PGCs)havethecapacitytoregeneratetotipotency

PGCsarenotpluripotent(ie donotcontributetochimaeras
wheninjectedintoblastocyst(summarisedinLeitchetal,2013)

PGCsexpresstranscriptionalnetworkrelatedtopluripotency (similartomESCs)

PGCscangiverisetopluripotentembryonicgermcells(EGcells)invitro

Mousedevelopment ES
blastocyst
E6.5
ICM
extraembryonic
ectoderm

TE

zygote epiblast
DNAdemethylation
Maintenanceofsome
chromatinmarks

DNAdemethylation
ReactivationofXi
Erasureofpolycomb
marks

PGCs
EG
E 10.511.5
Maturegametes

Page 14 of 124
 Stem cell derivation classic protocol

ICM LIF
ES cells
FCS/MEFs

Oct4 Sox2
Nanog Esg1

LIF
PGC
EG cells
FGF2,SCF
FCS/MEFs

Keysignallingpathwaysregulatingpluripotency(mESCs)

Stem cell derivation 2i protocol

(reaching the pluripotent ground state)

ICM LIF
ES cells
2i

Oct4 Sox2
Nanog Esg1

LIF LIF
PGC
2days EG cells
FGF2/SCF 2i
FCS/MEFs
Ying et al, 2008, Leitch et al, 2010

Page 15 of 124
 Invitromodels

EGvs EScomparison
ESderivedfromICM,EGderivedfromPGCs
lackofimprintsinEGcells(xEScells)
EGcellscaneraseimprintsuponfusionwithasomatic
cell
duetotheirgermline originEGcellsarebelievedtobe
globally(DNA)hypomethylated

Experimentaldesign

Leitch,McEwenetal,NSMB2013

Geneexpressionanalysis
(Affymetrix genearray)

Unsupervisedhierarchicalclustering

Thefirstprincipalcomponent(i.e.themajordifferenceinthesampleset)separatescelllines
maintainedin2i/LifandFCS/LIF/MEFs(FLM)(>2000genes,FDR<0.05,FC>1.5)

Thesecondandthirdprincipalcomponentsseparategender
Thethirdprincipalcomponentalsoseparatescelltype(EGvsES )(83genes)

Page 16 of 124
 2ivsclassiccultureconditions

SeealsoMarksetal,2012

2ivsclassiccultureconditions

Leitch,McEwenetal,NSMB2013

Mousedevelopment ES
blastocyst
E6.5
ICM
extraembryonic
ectoderm

TE

zygote epiblast
DNAdemethylation
Maintenanceofsome
chromatinmarks

DNAdemethylation
ReactivationofXi
Erasureofpolycomb
marks

PGCs
EG
E 10.511.5
Maturegametes

Page 17 of 124
 MeasuringDNAmethylationbyLCMS
method

LC/MS analysis
DNA enzymatic Deoxynucleosides
extraction digestion dT 5mdC
dA dC

dG 5hmdC

Agilent Triple Quadrupole

LC Atlantis C18 column 6490LC/MS

dC
Calibration curve dC
5hmdC

5mdC
Response

dG

fC

CaC
Concentration [fmol]

GlobalDNAhypomethylationin2i

Globallossof5mCin2iconditions
NodifferencebetweenESandEGcelllines(orbetweenmaleand
femalecells) male
Repeatsaswellassinglecopylociaffected(confirmedbybisulphite
analysis) Leitch,McEwenetal,NSMB2013

2ivsclassiccultureconditions
DNAmethylationlevelsarereversible

Genomewide5mClevelsfullyreversiblewithin5passages

Leitch,McEwenetal,NSMB2013

Page 18 of 124
 2ivsclassiccultureconditions
RegulationofDNAmethylation differentiation

DnmtTKOEScellsdieuponexitfrompluripotency
Dnmt3a/3bKOEScellsfailtodifferentiate

Leitch,McEwenetal,NSMB2013

Leitch,McEwenetal,NSMB2013

SummaryI

ESandEGcellsareverysimilaratthetranscriptionallevel,HOWEVER
Majortranscriptionaldifferencesfoundbetweenpluripotentcellsgrown
nFCS/LIFandin2iconditions

Nodifferenceingloballevelsof5mCbetweenESandEGcells,but2i
inducesgenomewideDNAhypomethylation (downregulation of
Dnmt3a,Dnmt3bandDnmt3l,nochangeinDnmt1!)

GlobalDNAmethylationlevelissimilarbetweenmouseICMandmouse
pluripotentESCsandEGCsgrownin2imedium

Page 19 of 124
 Leitch,McEwenetal,NSMB2013

EGs,genomicimprints&chimaeraformation

EGs,genomicimprints&chimaeraformation
EGcellslackgenomicimprinting(xEScells)
Pluripotent,contributetoallgermlayersinchimaeras
Classicderivationprotocol>highcontributionchimaeraslethal
(lackofgenomicimprinting)

(Tadaetal,1998)

Page 20 of 124
 Maintenanceofgenomicimprintsin2i

Maintenanceofgenomicimprintsin2iandchimaeracontribution

Leitch,McEwenetal,NSMB2013

EGs,genomicimprints&chimaeraformation

Leitch,McEwenetal,NSMB2013

Page 21 of 124
SummaryII

2iallowsforderivationofEGlineswithintactimprints(humanEGlines?)

2iderivedEGlinescangiverisetohealthyhighcontributionchimaeras

Neitherglobalhypomethylation norlackofimprintsaredistinguishingfeaturesofEGcells

Acknowledgment
lab members
Kirsten McEwen
Aleksandra Turp
Austin Smith (Wellcome Trust Centre for Stem
Buhe Nashun
Cell Biology, Cambridge)
Rachel Amouroux
Harry Leitch
Peter Hill
Billy Mansfield
TienChi Huang
Sarah Linnett
Anne Ferguson-Smith (University of Cambridge)

Bioinformatics
Tom Caroll
Gopu Dharmalingam

CSC Mass spec facility

Vesela Encheva
CSC genomics laboratory
Laurence Game

Page 22 of 124
 Biochemicalanalysisofearly
Mammaliangermcelldevelopment

NielsGeijsen
HubrechtInstitute

No conflict of interest to report

Learning aims:

Differences between germ cell specification in lower animals and mammals

A method for the in vitro generation of primordial germ cells from

pluripotent stem cells

Dazl, a essential factor for germ cell specification, suppresses mRNA

translation

Dazl targets mRNAs of pluripotency factors, differentiation mediators and

pro-apoptotic factors.

Page 23 of 124
Cellfatedeterminationthoughgermplasmlocalization

Vasa

Oskar

Gcl(Germcellless)

Nos(Nanos)

EarlyDrosophilaembryogenesis

Egg

Nucleardivisionwithoutcellcleavage

SyncytialBlastoderm(9nucleardivisions)

Nuclearmigration(4nucleardivisions)

Cellularblastoderm(6000cells)

Mislocalizationofgermplasmresultsinectopicgerm
cellformation

OskarRNA
Nosprotein

OskarProtein

VasProtein

NosRNA

Page 24 of 124
 Germplasmisconservedacrossmanyspecies

Drosophila Zebrafish Xenopus

ZhouandKing2004

Mammaliangermcelldevelopment

Blimp1GFP Stella/Dppa3
E7.25

Ohinataetal.,2008 MacLaren,2003

Oct4GFP Stella/Dppa3

E9.0 E9.5 E10.5 E11.5/E12.5

Langeetal.,2003
Molyneauxetal.,2001

Invitrogenerationofgermcellsfrom
embryonicstemcells

Science(2003)300:1251

Nature(2004)427:148

PNAS(2003)100:11457

Page 25 of 124
Modelingearlyembryonicdevelopment:Embryoidbodies

EScells HangingdropEBs

Modelingearlyembryonicdevelopment:Embryoidbodies

d2

d7

d14

Embryoid Bodies

Invitrogermdevelopmentofprimordialgermcells

ES cells:APextinction Day7 EB:APpositivecolony

Germcells

Hematopoieticcells
Parthenogenetic
DNMT1null

ESclones D4EG D7EG D10EG

clones clones clones
J1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Page 26 of 124
 AprimordialgermcellspecificDazlGFPreporter

+/+ Floxed
+/+ Tg
Dazl Chr 17 1 kb
NcoI NcoI
Exon 9 10 11

+/+
WT kb
gfp PZ
Floxed 7 Tg
+CRE recombinase 6 WT
5
Tg
gfp 4 Fl
probe

E12.5 E13.5
GFP expression

400
300
200
100
0
G G H Lv Lb B I
WT Dazl-GFP

InvitrogenerationofDazlGFPgermcells

80 Stella Dazl Stella Dazl

D12+

D16+

Stella-GFP
ES

ES+

ES

ES+

D7+

D9+

60
%GFP+

Dazl-GFP
Dazl
40 Aurkc
Asz1
CycT
20
Dmrt1
Fkbp6
Germ cell

0 Smc1b
0 2 4 6 8 10 12 14 Sohlh2
Day
Stag3
Stra8
Gtsf1
Comp.2 Taf7l
Mael
Rec8
Tdrd7
ES Dazl+ Tex19.1
Early PGC

Testis Stella
PGC Dnd1
Comp.1 Prdm1
ES Stella+
D12-16 Dazl+ Nanos3
D7-9 Stella+ Hprt
Ubiquitous

Mapk1
Pgk1
Ubc
0.25 4

Dazllocalizesincytoplasmicgranules(Pbodies)
Testis E12.5 In vitro PGC

GE-1
Dazl
10m DAPI

Page 27 of 124
 TheDazdomainisessentialforPbodylocalization

Dazl FL (1-298) RRM DAZ mCherry

Dazl C (1-165) RRM mCherry
Dazl N (121-298) DAZ mCherry

DazlFL-mCherry DazlC-mCherry DazlN-mCherry

5m

LossofDazlhasminimaleffectonPGGgeneexpression

Day 9 Day 12
Dazl knockdown

Asb9 Asb9

Dazl
Dazl

Control Control

LossofDazlhasminimaleffectonPGGgeneexpression

Page 28 of 124
Dazlassociateswithanetworkofubiquitouslyexpressed
RNAinteractingproteins

V5+R
Input

V5+I
IgG
PABP1

Igf2bp1

Elavl1

G3bp1

Dazl-GFP-V5

Rack1

-tubulin
IgG heavy chain

DazlcolocalizationwithIgfbp1andFragileXproteins

Igf2bp1 Fxr1 Fmrp

Dazl Dazl Dazl
DAPI DAPI DAPI

5 m

DazlbindsmRNAtranscriptsofkeypluripotencygenes

Exp. 1 Exp. 2
-GFP

-GFP
Input

Input
-V5

-V5
IgG

IgG

Sox2
Sall4

Zfp42

Suz12

Zic3
Oct4
Mvh
Tdrd7
Fmr1

Page 29 of 124
 Dazl -/- Dazl +/-

LossofDazlleadsto
aberrantexpression
ofpluripotency Sox2
DAPI

genes
Sall4
DAPI

Suz12
DAPI

Oct4
DAPI

Lin28
Ki67
DAPI

Dazlassociateswithanetworkofubiquitouslyexpressed
RNAinteractingproteins
V5+R
Input

V5+I
IgG

PABP1

Igf2bp1

Elavl1

G3bp1

Dazl-GFP-V5

Rack1

-tubulin
IgG heavy chain

FertilityphenotypeinFragileXmutantmice

WT Fxr1 cON

Page 30 of 124
 Fxr1hypomorphicmicephenocopyDazl/ mutation

Fxr1cON Control

Sox2 Sox2
DAPI DAPI

Suz12 Suz12
DAPI DAPI

Lin28 Lin28
Ki67 Ki67
DAPI DAPI

Dazlassociateswithtranscriptsofproapoptoticgenes

UV-IP Native IP Juv. Testis IP

PABP1
Input

Input

Input
GFP

GFP
GFP
IgG

IgG
V5

V5

Casp2
Casp7
Casp9
Pdcd7
Asb9

Dazlrecruitmentoftranslationalregulatorsto
specificmRNAtranscripts

Page 31 of 124
Acknowledgements
Geijsen group Netherlands Proteomics Center
Maaike welling Nikolai Mischerikov
Stefan van der Elst Javier Munoz
Nune Schelling Albert Heck
Diego DAstolfo
Nicolas Rivron
Manda Arbab BACPAC Resources Center, Oakland
Pieterjan Dierickx Pieter de Jong
Axel Beier Christine Jung
Javier Frias Aldeguer

Harvard Stem Cell Institute

May Chen NIRM
Christa Buecker
Donald Bloch NIH
Cody Tramp
Xinjie Chen NWO

Cold Spring Harbor Laboratories

Jie Wu
Adrian Krainer

Page 32 of 124
Pre-congress course
PCC13: Of stem cells and gametes: more
similarities than differences?

Current status of in vitro differentiation of

HPSC into female gametes

Susana M. Chuva de Sousa Lopes, PhD

Associate Professor
Dept. Anatomy and Embryology
Leiden University Medical Center
Leiden, The Netherlands

I have nothing to disclose

Learning objectives:

- What are HPSC (human pluripotent stem cells)?

- What are female gametes and why are they special?

- Why is there interest in generating female gametes from HPSC?

- What is the current state of in vitro differentiation to female gametes?

- What can you say in the clinic if you are asked about this topic?

Page 33 of 124
 What are HPSC (human pluripotent stem cells)?

Fertilized egg

Blastocyst (ICM)
Epigenetic
control??

Primordial
germ cells

Oocyte Sperm
Somatic Lineages
Germ cell lineage
Courtesy of Dr. K. Hayashi

How?

Epigenetic (re)programming as the possibility to be transcribed

A B C D E
heart

lung

muscle

gut

brain

Page 34 of 124
 Waddingtons epigenetic landscape

Tissue uniquely DNA hypomethylated regions: tissue barcode

What are MPSC (mouse pluripotent stem cells)?

Oocyte Sperm
Somatic Lineages
Germ cell lineage
Chuva de Sousa Lopes and Roelen, 2010

Page 35 of 124
 What are HPSC (human pluripotent stem cells)?

hESCs:

- EpiSC resemble human ESC

- Nave, ground, primed,..

OLeary et al, 2012

What are HPSC (human pluripotent stem cells)?

hiPSCs:

What are HPSC (human pluripotent stem cells)?

- In vitro differentiation (embryoid bodies, directed differentiation)

- Teratoma formation (tumor induction in mice)

- Chimera formation (integration of cells in embryo)

- Tetraploid complementation (generation of a complete embryo proper)

Oocyte Sperm
Somatic Lineages
Germ cell lineage

Page 36 of 124
What are HPSC (human pluripotent stem cells)?

What are female gametes and why are they special?

Hayashi and Surani, 2009

What are female gametes and why are they special?

Sasaki and Matsui, 2008

Page 37 of 124
 What are female gametes and why are they special?

- Mature oocyte is totipotent (zygote, blastomeres)

- Very scarce.a few mature each time

- Know how to reprogram:

- Spatial information to form an embryo
- Formation of extraembryonic tissues (placenta, amnion)

- Can be used to make patient-specific embryonic stem cell lines:

- Alternative to hiPSCs
- Regenerative medicine
- Drug testing (stem cells as the new patient)

- Used in assisted reproduction

Why is there interest in generating female gametes from

HPSC?
- Mature oocyte is totipotent (zygote, blastomeres)
- Very scarce.a few mature each time

- Know how to reprogram:

- Spatial information to form an embryo
- Formation of extraembryonic tissues (placenta, amnion)

- Can be used to make patient-specific embryonic stem cell lines:

- Alternative to hiPSCs
- Regenerative medicine
- Drug testing (stem cells as the new patient)

- Used in assisted reproduction:

- Infertility can result in serious psychological problems
- Unable to retrieve mature oocytes or gender issues
- Problems associated with gamete donation (legislation)
- Importance of the genetic link with the child

Current state of in vitro differentiation to female gametes?

Mouse:

Hubner et al., Science, 2003, 300:1251

Oct4GFP mESCs line
Differentiation in a monolayer
Expression of VASA after 12 days
Aggregates detached from colonies
Replated aggregates form follicle-like structures
Spontaneous activation of oocyte-like cells

Novak et al., StemCells, 2007, 24:1931

No evidence for meiosis !

Page 38 of 124
Current state of in vitro differentiation to female gametes?

Mouse:
Lacham-Kaplan et al., StemCells, 2006, 24:266
EBs cultured in mouse testis conditioned medium
After 6-7 days follicle-like structures
Oocyte-like cells 15-35m
No zona pellucida
Expression of several oocyte-markers
No evidence for meiosis!

Qing et al., Differentiation, 2007, 75:902

EBs cultured in DMEM+FCS
Germ cells observed in EBs after 4 days
Co-culture with granulosa cells from newborn mice
Expression of several oocyte-markers
No evidence for meiosis!

Current state of in vitro differentiation to female gametes?

Mouse:

Current state of in vitro differentiation to female gametes?

Mouse:

Nicholas et al., 2008

Page 39 of 124
Current state of in vitro differentiation to female gametes?

Mouse:

Current state of in vitro differentiation to female gametes?

Human:

Current state of in vitro differentiation to female gametes?

Human: Clark et al., HumMolGen, 2004, 13:727

EBs cultured in DMEM+FCS
Germ cells observed in EBs after 7-14 days
Expression of several oocyte-markers
No evidence for meiosis!

Chen et al., Hum Rep , 2006, 22:567

EBs cultured in DMEM+FCS
Germ cells observed in EBs after 7-14 days
Expression of several germ cell-markers
No evidence for meiosis!

Page 40 of 124
 Current state of in vitro differentiation to female gametes?

Human:

Duggal et al., 2014

Sun et al., 2014

What can you say in the clinic if you are asked about this topic?

Impossible!

References:

- Chuva de Sousa Lopes and Roelen, 2010, Differentiation 79:131

- OLeary et al., 2012, Nature Biotechnology 30:278
- Hayashi and Surani, 2009, Cell Stem Cell 4:493
- Sasaki and Matsui, 2008, Nature Reviews Genetics 9:129
- Hubner et al., 2003, Science, 2003 300:1251
- Novak et al., 2007, Stem Cells, 24:1931
- Lacham-Kaplan et al., StemCells, 2006, 24:266
- Qing et al., Differentiation, 2007, 75:902
- Nicholas et al., 2008, Human Molecular Genetics 18:4376
- Hayashi et al., 2012, Science 338:971
- Clark et al., 2004, Human Mololecular Genetics 13:727
- Chen et al., 2006, Human Reproduction 22:567
- Duggal et al., 2014, Veterinary Quaterly PMID 24593843
- Sun et al., 2014, Journal Genetics Genomics, 41:87

Page 41 of 124
Dept. Anatomy and Embryology, LUMC
Susana Chuva de Sousa Lopes
Lisbeth van Iperen
Yuvendran Muniandy
Michael Festens
Marijne Heeren
Sara Mendes
Maria Gomes Fernandes
Matthias Roost
Ana Melo Bernardo
Nannan He

Dept. Reproductive Medicine, UZ Gent

Petra De Sutter
Bjorn Heindryckx
Galbha Duggal
Sabitri Ghimire
Jasin Taelman
Sharat Warrier

IAP-Phase VII

Page 42 of 124
 ESHRE Annual Meeting 2014
Pre-Congress Course 13
Munich, Germany

In vitro differentiation of PSC into male gametes:

current status and the road ahead

Cristina Eguizabal, PhD

Stem Cell Therapy Unit
Basque Center for Blood Transfusion
and Human Tissues

Disclosure

Nothing to disclose
I have no commercial or financial relationships with
manufacturers of pharmaceuticals, laboratory supplies or
medical devices

Learning objectives
At the conclusion of this presentation, participants should be
able to:

Discuss pluripotent stem cells (PSC)

Explain generation of pluripotent stem cells
Why generation of gametes from PSC?
Explain generation of male germ cells from mouse pluripotent stem cells
Explain generation of male germ cells from human pluripotent stem cells
Conclusions and Future remarks

Page 43 of 124
Pluripotent Stem Cells (PSC) can be
obtained from cells located in the inner cell
mass of blastocysts (ESC), from primordial
germ cells (EGC) and from nuclear
reprogramming (SCNT and iPS)

The Nobel Prize in Physiology or Medicine

2012 was awarded jointly to Sir John B. Gurdon
and Shinya Yamanaka "for the discovery that
mature cells can be reprogrammed to become
pluripotent"

Sir John B. Gurdon Shinya Yamanaka

SCNT iPS

Page 44 of 124
Integrative delivery systems: Oct4 Klf4 INDUCED PLURIPOTENT STEM CELLS
cMyc
Retrovirus/lentivirus/linear Sox2 cMyc Non integrative delivery systems:
DNA/PiggyBac transposon Adenovirus/Sendai viral vectors/
Sox2 Oct4 episomal vectors/synthetic
Klf4 mRNA/proteins

Somatic cell
Cell, 2006

iPS GENERATION
Choice of starting cell types
Choice of factor delivery
Choice of number of factors

GonzlezF.etal.,2011

Lentivirus/retrovirus mediated reprogramming methods are still major

approaches for iPS generation

Why are we interested in the generation of germ cells in

vitro?

To study gametogenesis in vitro for a better understanding of this process

To study meiosis in vitro

To check the capability of PSC (ES/iPS) to form germ cells in vitro
PSC may constitute a future source of artificial gametes for clinical studies
and potential therapeutic applications
This system may provide a useful model for molecular genetic studies of
human germline formation.

Page 45 of 124
 IMPORTANT POINTS FOR HAVING MALE FUNCTIONAL GAMETES
DURING GERMLINE DIFFERENTIATION FROM PLURIPOTENT STEM
CELLS:
The sequence of in vivo events:

IMPORTANT POINTS FOR HAVING MALE FUNCTIONAL GAMETES

DURING GERMLINE DIFFERENTIATION FROM PLURIPOTENT STEM
CELLS:
VASA
The sequence of in vivo events:

Stra8

Scp3
H2AX

Acrosin

IMPORTANT POINTS FOR HAVING MALE FUNCTIONAL GAMETES

DURING GERMLINE DIFFERENTIATION FROM PLURIPOTENT STEM
CELLS:
The sequence of in vivo events:

Sertoli and Leydig cells

Growth factors
and hormones

Page 46 of 124
 IMPORTANT POINTS FOR HAVING MALE FUNCTIONAL GAMETES
DURING GERMLINE DIFFERENTIATION FROM PLURIPOTENT STEM
CELLS:
The sequence of in vivo events:

MEIOSIS

IMPORTANT POINTS FOR HAVING MALE FUNCTIONAL GAMETES

DURING GERMLINE DIFFERENTIATION FROM PLURIPOTENT STEM
CELLS:
H19 gene
The sequence of in vivo events: 0% methylated

75% methylated

100% methylated

MOUSE

Toyooka et al. PNAS 2003

Giejsen et al. Nature 2004
Nayernia et al. Developmental Cell 2006
Eguizabal et al. Differentiation 2009
Hayashi et al. Cell 2011
Peng et al. Biomed Res. Intl. 2013
Cai et al, BBRC 2013
Nakaki et al,. Nature 2013

Page 47 of 124
 Generation of male germ cells in vitro from mouse pluripotent
stem cells

Pluripotent stem cells used: ES, EG and iPS cells.

Sex of cell lines: XY and XX.
Transgenic reporters / overexpression genes used: MVH, Stra8, Prm1,
Stella, Blimp1, Prmd14 and Tfap2c.
Differentiation method used: EB formation and monolayer differentiation.
Culture conditions: FBS, BMP4, N2B27, Activin A, bFGF, SCF, Retinoic
Acid, Transferrin, Monothyoglicerol , Ascorbic Acid and Testosterone.
In vitro cells obtained: Epiblast, PGCs, SSCs and male haploid-like cells.
Epigenetic status of imprinted genes: correct
Functional assays: Transplantation of SSCs into sterile testis or ICSI.

PNAS, 2003

EB formation and co-culture system in presence of BMP4.

Transplantation of ESC aggregates to testicular capsule.
ESC derived cells can participate in spermatogenesis and
produce male germ cells in vitro.

NATURE, 2004

During embryoid body formation pluripotency

markers expression decreased with some
expression of male germ cell specific genes.
ICSI with haploid cells into recipient oocytes
EB microenvironment is permissive for male 50% of the embryos cleaved to 2 cell stage
germ cell development and meiotic 20% reach the blastocyst stage.
maturation, even though highly inefficient
FISH: normal for sex chromosomes

Page 48 of 124
 Dev Cell, 2006

Establishment of two cell lines (Spermatogonial

stem cells- SSCs) with patterns of differentiation
towards male germ cells . ICSI of in vitro-generated cells (haploid cells)
Derived from mESC (directed gene expression into oocytes of wildtype females.
for premeiotic and haploid male germ cells) Transfer of 65 embryos into the oviducts of
Formation of sperm with tail-like structures pseudopregnant females
Premature death (5 days to 5 months after
birth).
Abnormal methylation patterns and
phenotypic abnormalities

2009

Mouse EG and ES cells can form

in vitro PGCs via embryoid body
formation by adding RA

The expression pattern of

histone modification markers of
late PGCs (Prmt5 and
H3K27me3) is similar to in vivo
embryonic day E 11.5

When co-cultured with Chinese

hamster ovary (CHO) cells ,
some of the PGC enter meiosis
(SCP3 expression)

2011

Generation of in vitro primordial germ cell-like cells from

mES and miPS through epiblast-like cells having 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.

Page 49 of 124
 2013

Generation of male in vitro germ cells

by EB formation in presence of RA and
Testosterone.
iPSC derived cells can participate in
spermatogenesis and produce male
haploid-like cells in vitro.

2013

Generation of in vitro primordial germ cell-like cells from miPS

by EB formation in present of RA, FGF2 and SCF.
These PGCLCs express Integrin-b3 and SSEA1 markers of
spermatogonias.
Generation of in vivo ectopic reconstitution of seminiferous
tubules by transplantation of miPSCs with neonatal testicular
cells.

Direct EpiLCs and mESC conversion into primordial germ

cells like cells (PGCLCs) by over-expressing 3 TFs :
BLIMP1 (B), PRDM14 (P14) and TFAP2C (A)
Transfection of BVSCR26rtTA ES TF-PGCLCs characterization
cells with piggyBac transposon
expressing the TFs

More than 80% of the cells

expressed BV (Blimp1-
mVenus) and 30% of the
cells expressed SC (stella-
eGFP).

Simultaneous overexpression of 3 TFs directs EpiLCs, but not ESC, swifly

and efficiently into a PGC state
TFs showed faster kinetics of Blimp1 and Stella induction than the cytokines
TFs directly activate a PGC program
Normal offspring were obtained from TF-PGCLCs
NakakiF.etal.Nature(2013)

Page 50 of 124
 HUMAN

Bucay et al. Stem Cells 2008

PGCs
Tilgner et al. Stem Cells 2008; 2009
Park et al. Stem Cells 2009
Kee et al. Nature 2010
Panula et al. HMG 2011
Eguizabal et al. Stem Cells 2011
SSCs/haploid-like cells
Medrano et al. Stem Cells 2011
Schatten et al. Cell Reports 2012
Ramathal et al. Cell Reports 2014

Generation of germ cells in vitro from human pluripotent stem cells

Pluripotent stem cells used: ES and iPS cells.

Sex of cell lines: XY and XX.
Transgenic reporters / overexpression genes used: DAZ, DAZL, BOULE
and VASA.
Differentiation method used: EB formation and monolayer differentiation.
Culture conditions: FBS, BMP4, -7, -8b, Activin A, bFGF, hLIF, Retinoic Acid,
R115866, Nicotinamide, Transferrin, Insulin, Selenium, Monothyoglicerol and
Ascorbic Acid.
In vitro cells obtained: PGCs, Putative Sertoli, SSCs and male haploid-like
cells
Epigenetic status of imprinted genes: correct

2008

Generation a novel protocol based on the expression of CXCR4 receptor

during the in vitro differentiation of PGCs from hESC.
This differentiation process is accompanied by the development of putative
Sertoli-like cells.

Page 51 of 124
 2008

Generation a novel protocol (EBs + BMP4) for differentiating in vitro PGCs

from hESC.
These PGCs expressed specific germ cell markers and showed a removal of
the parental imprints and chromating modification changes.

2010

The generation and characterization of human embryonic stem cell lines stably
carrying a VASA-pEGFP-1 reporter construct that expresses GFP in a population of
differentiating human embryonic stem cells 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.

Page 52 of 124
 Nature, 2009

Generation of in vitro PGC from male and female hESC

DAZL, DAZ and BOULE promote PGC formation and germ-cell
progression to meiosis and formation of haploid germ cells that
ressemble round spermatids

2011

iPSC can form in vitro meiotic and post-meiotic haploid cells

over-expressing DAZ, DAZL and BOULE.

2011

Generation of a novel differentiating 2 step-protocol without

overexpressing any genes.
iPS can form in vitro meiotic and haploid like-cells.
The post-meiotic-like cells generated have features of human
spermatids expressing Acrosin.

Page 53 of 124
 Eguizabal et al. Stem Cells, 2011

Characterization of hiPS cell lines

Oct4 SSEA-4 Dapi Merged

KiPS2

CBiPS2

Nanog Tra-1-81 Vasa Dapi Merged

KiPS2

CBiPS2

Human
Testis

Eguizabal et al. Stem Cells, 2011

Timeline of in vitro differentiation of human iPS cells as a model for

derivation of postmeiotic germ cells

R115866

Eguizabal et al. Stem Cells, 2011

Page 54 of 124
Characterization of human germ-like cells after enrichment by FACS sorting at
6 weeks of culture

CD49f/CD9: markers of SSCs

CD90/SSEA4: markers of ES cells

Eguizabal et al. Stem Cells, 2011

Differentiation towards human germ-like cells at 6 weeks of culture

Germ cell marker: VASA

Sertoli cell markers: Vimentin and Nestin
Leydig cell marker: 3-HSD
Eguizabal et al. Stem Cells, 2011

Meiotic progression of germ-like cells at 9 weeks of culture

Eguizabal et al. Stem Cells, 2011

Page 55 of 124
 Haploid formation of germ-like cells at 10 weeks culture

0.43-2.33% haploid-like cells in presence of R115866

Eguizabal et al. Stem Cells, 2011

Methylation status of H19 promoter DMR were analyzed by bisulphite

sequencing in undifferentiated hiPS and hES, and after 6 and 10 weeks of
differentiation

H19: maternally gene

expressed and paternally
0% methylated
gene methylated
75% methylated

100% methylated
Eguizabal C et al. Stem Cells, 2011

2011

Generation of in vitro PGCs

from hESC and hiPS over-
expressing VASA and/or DAZL.
The hES/hiPS can form
correctly in vitro meiotic and
post-meiotic male haploid cells
over-expressing VASA and/or
DAZL in 14 days.
The in vitro germ-like cells over-
expressing only VASA present
the typical pattern of methylation
status for H19 as the normal
germ cells.

Page 56 of 124
 2012
hESCs and iPSCs cultured in
SSC conditions differentiate
2012 directly into advanced male
germ cell lineages including
postmeiotic,spermatid-like cells
in vitro without genetic
manipulation (10 days)

hPSCs differentiated in SSC

culture conditions exhibit haploid
features

Differentiation of hPSCs in
SSC culture yields cells that
express markers of
spermatogonia, spermatocytes
and spermatids

2014

all iPSCs that exited tubules formed

primitive tumors.
iPSCs with AZF deletions produced
significantly fewer GCLCs in vivo with
distinct defects in gene expression
xenotransplantation of human
iPSCs directs germ cell
differentiation in a manner
dependent on donor genetic status

Conclusions and Future remarks

Early germ cells and functional sperm have been obtained through
mouse ESC and iPS using 2 step protocols (in vitro and in vivo).

By using fully in vitro protocol for the generation of mouse male germ
cells, the methylation pattern and offspring are abnormal.

Normal healthy offspring with normal methylation patterns of imprinted

genes if gametogenesis is resumed in in vivo conditions.

Further studies will be necessary to develop efficient protocols to get in

vitro germ cells.

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.

Page 57 of 124
 Anna Veiga
Juan Carlos Izpisua-Belmonte Fanny Vidal
Nuria Montserrat Lydia Garca-Quevedo
Rita Vassena
Montserrat Barragan
Elena Garreta
Alessandra Giorgetti

Basque Center for Blood Transfusion and Human Tissues

[email protected]

THANKS FOR YOUR ATTENTION!!!

Page 58 of 124
 Functional Characterization of Adult Ovary-derived
Oogonial Stem Cells in Mice, Monkeys and Women
Jonathan L. Tilly, Ph.D.

Professor and Chair

Department of Biology
Laboratory of Aging and Infertility Research
Northeastern University
Boston, Massachusetts 02115, USA

[email protected]

Presented at the ESHRE Pre-congress Course 13 (SIG Stem Cells) , Munich, Germany 29 June 2014

DISCLOSURES

Jonathan L. Tilly, Ph.D., declares the following:

interest in intellectual property described in U.S. Patents

7,195,775
7,850,984
7,955,846
8,642,329
8,647,869
8,652,840

interest in intellectual property described in U.S. Patent Applications

11/131,152
11/131,153
61/502,840
61/885,559
61/887,569
PCT US 2014/032010

and,

interest as a scientific co-founder of OvaScience, Inc.

(Cambridge, MA; www.ovascience.com)

LEARNING OBJECTIVES

At the conclusion of this lecture, the participant:

1. Will be introduced to published studies regarding the identification,

isolation and characterization of female germline or oogonial (oocyte-
producing) stem cells (OSCs) in adult mammalian ovaries

2. Will be better informed of studies questioning the existence of OSCs,

as well as the conceptual and experimental limitations of these studies
which led to the conclusion drawn that OSCs do not exist

3. Will be able to integrate currently available evidence on the properties

of OSCs in mice, monkeys and women into discussions of how
regenerative medicine-based technologies involving OSCs could
provide new tools to combat aging related infertility and menopause

Page 59 of 124
 The Female
Biological Clock
Female New
Germline or Oocytes
Oogonial Stem
Cells (OSCs) Fertility &
Ovarian
Function
Ovarian
Reserve
Oocyte loss due to Insults
natural causes
MORE
Oocytes Lost
(Cell Death)

Infertility
Menopause Accelerated Onset

= OOCYTE RESERVE NOT FIXED AT BIRTH (OVARIES TESTES)

Independent Confirmation (mouse studies):

Zou et al. Nature Cell Biology 2009 12: 631636

OSCs isolated from juvenile and adult ovaries
Transplanted OSCs produce eggs that fertilize and yield viable offspring

Pacchiarotti et al. Differentiation 2010 79: 159170

OSCs isolated from neonatal and adult ovaries
Cultured OSCs generate oocytes that form follicles in vitro

Live Cells: FACS

Purification of AND,
if one still feels strongly against using
OSCs based on cell Ddx4 antibodies to purify OSCs,
surface expression of the antibodies against Ifitm3 (Fragilis), a
well accepted transmembrane protein
germ cell-specific protein, in germ cells, workDdx4+
equally well
Ddx4 (Vasa, Mvh)
intracellular extracellular
Tilly lab, unpublished data
In oocytes, Ddx4 is Ddx4
FACS
C-terminal
localized exclusively within protein
OSC plasma
NH 2 COOH Ddx4 Antibody
membrane
the cytoplasm
OSC plasma NH2
In OSCs, the C-terminus of membrane
Ifitm3 Ifitm3
Ddx4 is exposed on the protein Antibody
outer plasma membrane
Target this externalized COOH

epitope with a C-terminal MACS

Ddx4-specific antibody for Stem Cell Dev 2011 20: 21972204
Live Cells: MACS
live cell sorting
Nature Cell Biology 2009 11: 631636
Nature Medicine 2012 18: 413421
Nature Protocols 2013 8: 966988

Page 60 of 124
 Why is this so important? Because at least one person has
disputed publications from us and others based on his own
labs inability to viably sort OSCs using Ddx4 antibodies

Liu in Sweden says that he initially believed Wus paper

when it came out. But his group could not repeat the
technique [of Ddx4/Vasa antibody-based sorting]. To
bypass the cell-surface problem with vasa, Liu used an
approach that tracks the protein inside the cells.

The Liu lab dissociates ovaries for analysis using trypsin!

our results show that no mitotically active

Ddx4-expressing female germline progenitors
exist in postnatal mouse ovaries.

Collect ovaries at
day 8, disperse,
filter (40-m pores)
and culture cells

Ddx4Cre;Rosa26rbw/+
Proliferation of RFP-positive (Ddx4-Cre recombined) cells in
Ddx4 promoter activation = just those
cells switch from GFP to RFP/YFP/CFP crudely dispersed ovarian cell preparations was not observed
(Cre-based recombination) over short term culture (24, 48, 72 hours) = OSCs do not exist

Limitations and Caveats:

No control experiments presented to document germ cell-
specificity of Cre expression (no leakiness)
Even if specific for germ cells, Ddx4-Cre approach
recombines all germ cells equally, and thus cells identified
+ as positive by RFP expression could be oocytes, OSCs or
any cell that has activated the Ddx4 promoter fragment =
identity confirmation a must, but no effort was made to
Ddx4Cre Rosa26rbw/+ show that the cells studied are OSCs and not oocytes!
Ubiquitous GFP expression

2012 PNAS study from Liu and colleagues: a bold

conclusion from inconclusive data the major flaws
Ovaries dispersed exactly as described
Ddx4-Cre; by Liu and colleagues (trypsin-based
Rosa26tdTm/ Rosa26tdTm/+
+ disruption, filtered through 40-m cell
strainer to obtain cells studied)

Collect
ovaries

Simple PCR-based screen of

Ddx4-Cre;Rosa26tdTm/+ this fraction (unclear why not
done by Liu and colleagues)
Ddx4 promoter activation = just those
cells activate tomato red expression
(Cre-based recombination)
MAJOR FLAW #2:
Crude cell fraction containing Ddx4-
expressing cells studied by Liu and
colleagues is contaminated with Ddx4-
MAJOR FLAW #1: expressing oocytes, which are RFP-positive
Ddx4 promoter-driven but incapable of proliferation!
fluorescence pattern is
widespread in ovaries,
which is NOT consistent
+ with germ cell-specific
expression of the
Oocyte-
specific
reporter = leaky genes
promoter!
Ddx4Cre Rosa26tdTm/+
No reporter expression

Page 61 of 124
 If used the correct way, the Ddx4-Cre mouse
provides clear experimental evidence that
mitotically active female germline progenitors
exist in postnatal mouse ovaries

Ddx4-Cre; Ovaries dispersed and sorted for:

Rosa26tdTm/+ Rosa26tdTm/+ Tomato red
ecDdx4 (C-terminal antibody, APC)

Collect Tomato+

ovaries

Ddx4+

Tomato red
Tomato+

Ddx4-Cre;Rosa26tdTm/+
Ddx4 promoter activation = just those
cells activate tomato red expression
(Cre-based recombination)
Collect ecDdx4/Tomato Red dual-positive
cells and plate in culture: germ cell colony
establishment and proliferation
APC (Ddx4)

+
Ddx4Cre Rosa26tdTm/+
No reporter expression

X
X
BOTH studies based on circumstantial and indirect negative data

NEITHER study even attempted to reproduce what we and others have done
by simply isolating the cells they claim do not exist using validated and
detailed antibody-based sorting protocols why not?

None of this should not be acceptable at this stage if one wishes to refute the
findings of others and us, and claim that OSCs do not exist

Evolutionary conservation of
Annu Rev Genet 2005 39: 173195
female germline stem cells Cell Res 2007 17: 1525

Science 2010 328: 15611563

Mice
Nature 2004 428: 145150
Rats
Cell 2005 122: 303315
Giraffes
Nature Cell Biology 2009 11: 631636
Differentiation 2010 79: 159170
J MolCows
Cell Biol 2011 3: 132141
Baboons
Stem Cells Dev 2011 20: 21972204
Nature Medicine 2012 18: 413421
Rhesus Macaques*

*NIH trial ongoing

Nature Medicine 2012 18: 413421

Page 62 of 124
 Transplanted mouse OSCs carrying a traceable
transgene generate functional oocytes in vivo**
Genotype analysis of pups delivered by a wild type female mouse, transplanted at 2
months of age with OSCs isolated from ovaries of young adult TgOG2 (PEOct4-Gfp)
transgenic female mice, confirms delivery of offspring carrying the transgene reporter
(**independent confirmation of mouse OSC transplantation data reported by others:
Nature Cell Biology 2009 11: 631636 and J Mol Cell Biol 2011 3: 132141)

Offspring
Size TgOG2 Wild type
Marker (+) () #1 (F) #2 (F) #3 (M)

TgOG2 transgene

Wild type female 22919 (intraovarian transplant of TgOG2 OSCs) mated with wild type male:
2 pups (1 female, 1 male) negative for TgOG2 transgene (host oocytes)
1 female pup positive for TgOG2 transgene (TgOG2 OSC-derived oocyte)

Four breeding pairs have produced 38 offspring,

6 of which carry the TgOG2 transgene (15.8%)

In-vivo OSC function in adult female Rhesus macaques

Wolff EF, Libfraind LL, Weitzel P, Woods DC, Feng Y, Tilly JL, DeCherney AH, Tisdale JF. Oogonial stem
cells generate mature oocytes in an autologous Rhesus macaque transplantation model. Reproductive
Sciences 2014; 21 (Supplement): 119A

Bright-field GFP

OSC-derived MII Oocyte*

(*fertilized, 2PN embryo)

Remove one ovary, 2 3

purify OSCs for in Expand GFP retrovirus
vitro culture

The Female
Biological Clock
OSCs New
Oocytes

If we label new Fertility &

oocytes as they are Ovarian
being made, what is Function
their natural fate? Ovarian
Reserve
Oocyte loss due to Insults
natural causes
MORE
Oocytes Lost
(Cell Death)

Infertility
Menopause Accelerated Onset

Page 63 of 124
 Lineage tracing to map the in-vivo physiological fate of
newly generated oocytes in adult ovaries
Stemcell DifferentiatingCell TerminalCell
(meioticentry)
Stra8 expression
sperm
MALE SSC

Prenatal: PGC
FEMALE Postnatal: OSC
oocyte

Stra8-LSL-YFP Stra8 rtTA +Doxycycline Stra8 rtTA

TRE Cre TRE Cre
transgenic mouse x
Rosa26 stop YFP Rosa26 YFP

Stimulated by retinoic acid

gene 8 or Stra8:
Germ cell specific
Initiates meiotic commitment
Stra8 gene knockout = no oocytes Recombined cells Recombined cells
or spermatozoa produced (gonadal are permanently are fluorescent
dysgenesis and sterility)
Nature Genet 2006 38: 14301438
marked at the (YFP-expressing)
Proc Natl Acad Sci USA 2008 105: 1497614980 genomic level

Lineage tracing to map the in-vivo physiological fate of

newly generated oocytes in adult ovaries

Postnatal Postnatally-derived
Induction (adult oocytes are used to
life) = make babies?

Endowed oocyte Mosaic oocyte pool in Mosaic litter?

pool at birth adult ovary?

How long do oocytes produced during fetal

Prenatal
development (viz. endowment at birth, all
Induction (fetal
YFP+) persist in adult ovaries?
life)

What does the future hold for mammalian OSCs?

Impact on womens reproductive health**
6
Isolation of OSCs from
Diagnostics: egg quality,
neonatal, juvenile, or adult
ovarian reserve, etc.
human ovarian cortex
1
Cryopreserve and bank 5
for future use without or Identification of
with ex vivo expansion OSC activators

2 4
Adjunct to IVF: AUGMENT Generate new oocytes
procedure (autologous to restore or enhance
mitochondrial transfer) natural fertility
3
In-vitro maturation
of OSC-derived
oocytes into eggs

Bioenergetic enhancers Return expanded OSCs

(a.k.a. egg vitamins) to ovaries (technology 1)
Provide therapeutic OSC
mitochondrial numbers
activators (technology 5)
mitochondrial activity

**Commercial isolation and use of OSCs for these and other purposes are protected by several issued patents and patent applications
licensed exclusively to OvScience: U.S. Patents 7,955,846, 8,642,329, 8,647,869 and 8,652,840; U.S. Patent Applications 11/131,153,
11/131,152, 61/502,840, 61/885,559, 61/887,569 and PCT US 2014/032010)

Page 64 of 124
Heterologous ooplasmic transfer:
reinvigoration of energetically
compromised eggs through
donor mitochondria?

Human Reproduction Vol.16, No.3 pp. 513-516, 2001

BRIEF COMMUNICATION

Mitochondria in human offspring derived from ooplasmic

transplantation
Jason A. Barritt, Carol A. Brenner, Henry E. Malter and Jacques Cohen

Heterologous ooplasmic transfer:

reinvigoration of energetically compromised eggs
through female germline mitochondrial transfer at ICSI?

Transfer of mitochondria from No. of Success

Pregnancies Live Births
donor eggs Cycles Rate

Cohen et al., 1997, 1998; Brenner et

30 13 16 43%
al., 2000; Barritt et al., 2000, 2001

Dale et al., 2001 1 1 2 100%

Lanzendorf et al., 1999 4 1 2 25%

Huang et al., 1999 9 4 5 44%

44 attempts, 19 pregnancies achieved (25 babies): 43% success rates!

The promise and perils of heterologous (non-patient

matched) ooplasmic transfer
Ooplasm

= mitochondrion,
makes ATP as a
source of energy
for the egg

Egg to be
fertilized ATPATP
Donor egg from
young woman ATP

Insufficient Energy
Sufficient Energy
Halted by FDA
in 2002
SUCCESSFUL
NO fertilization orfertilization
POOR embryo and
embryo development
development = = Heteroplasmy
PREGNANCY
FAILED pregnancy 3 genetic parents
Consequences?
Foreign gene transfer

Page 65 of 124
 Could we use Human OSCs Human Ovarian Somatic Cell
autologous OSC
mitochondria?
Perinuclear localization Perinuclear localization Diffuse
cytoplasmic
Similar to other stem cell localization

types
Mitotracker Mitotracker

Mitochondrial DNA OSC Soma

Subject 1
(mtDNA) mutations Soma ve
Intact
Intact
Accumulate with age in mtDNA mtDNA4977 (internal
somatic cells primers)
mtDNA
1 16569

mtDNA4977 Subject 2
8470 - 13447 OSC Soma
Soma ve
Intact
Intact
OSCs free of common mtDNA mtDNA4977
(internal
deletion mutation primers)

Electron microscopic evaluation of mitochondrial

ultrastructure in oogonial stem cells
Very small, with electron dense matrices and few cristae, closely
matching ultrastructural features of mitochondria in oocytes
(very different from mitochondria in somatic cells)

OSCs Soma

Mitochondria present in human OSCs exhibit the highest

energetic capacity compared with other human cell types

1.4
pmol ATP produced per fg mtDNA

Oogonial stem cells

1.2 Mesenchymal stem cells
Ovarian somatic cells
1.0 Embryonic stem cells
induced pluripotent stem cells
0.8

0.6

0.4

0.2

0.0
10 15 20 30

Time (minutes)

Page 66 of 124
 Improving IVF success with OSC-based technology

Poor in-vitro fertilization (IVF) AUGMENT**

success rates in women of (Autologous Germline Mitochondrial Energy Transfer)

advanced maternal age reflect

unmet energy needs in the egg Isolate Mitochondria

Inject mitochondria
= mitochondrion, at time of ICSI
makes ATP (energy
OSCs
for the egg)

Egg to be
fertilized
ATP From
Egg to be
fertilized
ATP
same
patient

Insufficient Energy Sufficient Energy

NO fertilization or POOR embryo SUCCESSFUL fertilization and

development = embryo development =
FAILED pregnancy HEALTHY pregnancy

**AUGMENT is detailed in U.S. Patents 8,642,329 and 8,647,869

What does the future hold for mammalian OSCs?

Impact on womens reproductive health**
6
Isolation of OSCs from
Diagnostics: egg quality,
neonatal, juvenile, or adult
ovarian reserve, etc.
human ovarian cortex
1
Cryopreserve and bank 5
for future use without or Identification of
with ex vivo expansion OSC activators

2 4
Adjunct to IVF: AUGMENT Generate new oocytes
procedure (autologous to restore or enhance
mitochondrial transfer) natural fertility
3
In-vitro maturation
of OSC-derived
oocytes into eggs

Bioenergetic enhancers Return expanded OSCs

(a.k.a. egg vitamins) to ovaries (technology 1)
Provide therapeutic OSC
mitochondrial numbers
activators (technology 5)
mitochondrial activity

**Commercial isolation and use of OSCs for these and other purposes are protected by several issued patents and patent applications
licensed exclusively to OvScience: U.S. Patents 7,955,846, 8,642,329, 8,647,869 and 8,652,840; U.S. Patent Applications 11/131,153,
11/131,152, 61/502,840, 61/885,559, 61/887,569 and PCT US 2014/032010)

Woods DC, Tilly JL. Germline stem cells in adult mammalian

ovaries. In: Ten Critical Topics in Reproductive Medicine, S.
Sanders, editor; Science/AAAS, Washington, DC 2013, pp. 1012

In vitro oogenesis: screening for factors that drive

OSCs into oocytes and eggs (oogenic activators)

Oogenic activators identified thus far in OSCs:

Bone morphogenetic protein 4 (BMP4): see Fertil Steril 2013 100: 14681475
Estrogen acting via its alpha-receptor isoform (blocked by progesterone)
White adipocyte-derived growth factor (as-yet unidentified)

Page 67 of 124
 What does the future hold for mammalian OSCs?
Impact on womens reproductive health**
6
Isolation of OSCs from
Diagnostics: egg quality,
neonatal, juvenile, or adult
ovarian reserve, etc.
human ovarian cortex
1
Cryopreserve and bank 5
for future use without or Identification of
with ex vivo expansion OSC activators

2 4
Adjunct to IVF: AUGMENT Generate new oocytes
procedure (autologous to restore or enhance
mitochondrial transfer) natural fertility
3
In-vitro maturation
of OSC-derived
oocytes into eggs

Bioenergetic enhancers Return expanded OSCs

(a.k.a. egg vitamins) to ovaries (technology 1)
Provide therapeutic OSC
mitochondrial numbers
activators (technology 5)
mitochondrial activity

**Commercial isolation and use of OSCs for these and other purposes are protected by several issued patents and patent applications
licensed exclusively to OvScience: U.S. Patents 7,955,846, 8,642,329, 8,647,869 and 8,652,840; U.S. Patent Applications 11/131,153,
11/131,152, 61/502,840, 61/885,559, 61/887,569 and PCT US 2014/032010)

Woods DC, Tilly JL. Germline stem cells in adult mammalian

ovaries. In: Ten Critical Topics in Reproductive Medicine, S.
Sanders, editor; Science/AAAS, Washington, DC 2013, pp. 1012

In vitro egg formation from purified OSCs: is it feasible and

how do we do accomplish it?

Preantral
Nature Medicine follicle
Human Oocyte
2012 18: 413421 Primary
follicle Development

Antral
follicle
Primordial
follicle

Human Reproduction 2008

23: 11511158
Semin Reprod Med 2011
29: 1523

inject
Engineer to
express GFP

Oogonial stem cells Oocyte

GFP-positive egg?
Human ovary tissue Chromosomal
analyses and

DDX4-Ab
X
= developmental
competency?
FACS

Page 68 of 124
If OSC-based technologies can be developed and validated
for clinical management of female fertility, are OSCs always
present in ovaries to isolate and work with?

Mouse Human
(% of viable cells sorted)
OSC Yield Per Ovary

YES: OSCs are still present in ovaries after

age-related ovarian failure
Can we re-activate OSCs in ovaries that have failed?
Can we derive fully functional eggs from OSCs ex vivo?

The results presented to you today are even more compelling to consider if
the outcomes observed by us and others over the past decade of work are
actually due to the functional properties of cells that do not exist

The Female
Biological Clock
OSCs New
Oocytes

Fertility &
Ovarian
Function
Ovarian
Reserve
Oocyte loss due to Insults
natural causes
MORE
Oocytes Lost
(Cell Death)
Sustain quality of life
in women as they age?

Infertility
Menopause Accelerated Onset

Page 69 of 124
 If we delay ovarian failure, A B C
females age better!!
Nature Genetics 1999 21: 200203

aged female mice

Normal
No mouseopause

skin wrinkling deafness I look (and feel) marvelous, and Im

muscle cancer free!
cataracts
fat
hair loss

cognitive
deficits

Bone
density Normal No mouseopause

Ovarian replacement therapy can dramatically improve the quality of life

in aging females (Proc Natl Acad Sci USA 2007 104: 52295234)

THANKS
Laboratory
(past and present)
Ning Wang Eun-Sil Park
Yvonne White Cleo Szmygiel
Lek Satirapod Deanna Navaroli
Collaborators Yuichi Niikura Kshama Chandrasekhar
Anthony Imudia
Northeastern University Joshua Johnson
Dori Woods
Financial Support
Saitama University
Yasushi Takai NIH R37-AG012279
Hiroyuki Seki NIH R21-HD072280
Osamu Ishihara NIH F32-AG034809
Glenn Foundation
University of Edinburgh
Evelyn Telfer
Richard Anderson

NICHD, NIH
Erin Wolff
John Tisdale
Alan DeCherney

Page 70 of 124
 Domitoticallyactivefemalegermline
progenitorsexistinpostnatalmouseovaries?

Kui Liu
DepartmentofChemistryandMolecularBiology
UniversityofGothenburg,Sweden

Noconflictofinterests

Learningobjectives

Tolearnthecurrentunderstandings
ofthefemalegermlinestemcells:
iftheyexist,andiftheyarefunctinal.

Page 71 of 124
 Outline
Postnataloogenesis:atopicunderdiscussion.

Femalegermlinestemcellsinadultovary?The
issuesofisolatedcells.

Ourowndata:nomitoticallyactiveDdx4(Vasa)
positivefemalegermlineprogenitorsinpostnatal
ovaries.

Challenging the classic principle of female reproduction:

Follicular renewal in adult ovaries

Folliclecounting
Mathematicmodel

Johnsonetal.andTillyJL
Nature,2004

BMandPeripheralblood
Transplantation

Johnsonetal.andTillyJL
Cell,2005

Follicularrenewalinadultovaries:77folliclesperday?

Proliferated
germcell? Renewal
follicles?

Ovarianfolliclesarerecovered1day
afterbonemarrowtransplantation

Johnsonetal.andTillyJL, Johnsonetal.andTillyJL,
Nature,2004 Cell,2005

Page 72 of 124
 Similar experimental approaches showed
no follicular renewal in adult ovaries

Folliclecounting BristolGouldetal.,DevBiol.2005
Mathematicmodel

Folliclecounting KerrJBetal.,Reproduction.2012

Similar experimental approaches showed

no follicular renewal in adult ovaries

Transplantationand
Egganetal.,Nature.2005
Parabioticmousemodels

Transplantation BegumSetal.,HumReprod.2008

TransplantingGFPpositivebonemarrowintoSCIDmice
neverleadtoanyGFPoocytes.
Unpublisheddata,KuiLiugroup

A B
GFP-expressing bone marrow cells from Rainbow/+ females were transplanted into
adult SCID females through tail vein injection. No fluorescent oocyte was observed in
the ovary of recipients after 3 months of injection.

Page 73 of 124
Challenging the classic principle of female reproduction:
Female germline stem cells in postnatal ovaries

Zouetal.andJiWu,NatCellBiol,2009

Whiteetal.andTillyJL,NatMed,2012

Female germline stem cells isolated from postnatal

mouse ovaries by DDX4 (Vasa) antibody

Thecellsweresortedbymagnetic
activatedcellsorting,bytheuseofthe
DDX4 (Mvh,orVasa)antibody.

Mousefemalegermlinestemcells

Zouetal.andJiWu,NatCellBiol,2009

Oogonial stem cells isolated from postnatal mouse

and human ovaries by DDX4 antibody

Mouseoogoniastemcells
Thecellsweresortedby
Fluorescenceactivatedcellsorting
whichisalsobytheuseoftheDDX4
antibody.

Whiteetal.andTillyJL,NatMed,2012
Humanoogoniastemcells

Page 74 of 124
 Theisolationmethods

TheDdx4proteinslocateincytoplasmofmousegermcells

Ddx4

PI

Ddx4isamemberoftheDEADboxproteinfamilywhichlocatedincytoplasm
ofgermcells.

Toyookaetal.,MechDev,2000

Transmembrane domain in Ddx4 (Mvh) protein?

Zouetal.andJiWu,NatCellBiol,2009

TillyJLandTelferEE, MolHumReprod,2009

Page 75 of 124
NotransmembranedomaininDdx4proteinaftertheTMpredprediction

Onlyscores
above2844 are
considered
significant.

AnalysisbyKui
Liugroup.

http://www.ch.embnet.org/software/TMPRED_form.html

http://www.ch.embnet.org/software/TMPRED_form.html

Page 76 of 124
Did different groups independently repeat the isolation
of female germline stem cells?

WoodsDC,WhiteYA,TillyJL.ReprodSci.2013

Nofunctionaloocytesreported
Oct4reportercells JPacchiarottietal.,Differentiation,2010

Nottestedforfunctions

Culturedmixedovariancells CaoHetal.,PLoSONE,2012

DifferencesbetweencellsfromWus
groupandTillys group

1. Cellsizes.
2. Proliferationrateinvitro.
3. Characteristicsofthesecellsinculture.
4. Livepupsobtainedornot?

Whiteetal.andTillyJL,Nature,2012
TheFACSisolatedmouseovariancellsaresignificantly
smallerthannaturalPGCs

8m

Isolatedmouse Ddx4
ovariancells

Brdu

8m

Isolatedhuman Ddx4
ovariancells

Brdu

Bar:30m

Page 77 of 124
 Johnsonetal.andTillyJL,Nature,2004
Thesizeofputativemousefemalegermlinestemcellsinovaryis
similarwiththesizeofoocyteinprimordialfollicle

FGSCs? FGSCs?
Ddx4

Oocyteinprimordial
Brdu
follicle(about10um)

NoBarinFigure

ProliferatedDdx4positivecellsinovary:biggerthan10uM?

Zouetal.andJiWu,NatCellBiol,2009
Thesizeofputativemousefemalegermlinestemcellsinovaryis
similarwiththesizeofoocyteinprimordialfollicle

Bar:25m

isolatedDdx4positivecellsinmouseovarybyDdx4
antibody:12to20m

TheisolatedcellsfromJiWuandJTillylabsaresignificantlydifferent

Cellsize:1220m
Wu
Proliferation:Starting in24hafter
MACS seeding
isolated
cells Colonyformationinculture:Never
formcolonyindish.

Cellsize:8m
Tilly
FACS Proliferationrate:Veryslowinprimary
isolated culture(nodetailsreported)
cells
Colonyformationinculture:Colony
formationafter45weeks ofculture.

Page 78 of 124
FunctionalidentifyofOSCsfromdifferentgroup

Groups Developmental Stages References

Ji Wus group Live pups Zou et al., 2009, Nat Cell

Biol.
J Tillys group Blastocyst White et al., 2012, Nat
Med.
Izadyars group Oocyte-like cells Pacchiarotti et al., 2010,
Differentiation.
JL Huas group No functional study Cao et al., 2012, PLoS
One

Live pups are only obtained from the cells

derived from Ji Wus group.

DothetransplantedGFPpositivestemcellsmigratein
donorovaries?

Thediffuselocationoftransplantedcellsafterinjection.

Zouetal.andJiWu,NatCellBiol,2009

Theinjectedcellshavenocapabilitytomigrateinwhole
ovary.

Injected
cells

TheinjectedGFPcells(arrow)onlypresentaroundthesiteofinjection.

UnpublisheddataofKuiLiugroup

Page 79 of 124
 Anonmanipulatedsystem:Rosa26rbw/+;Ddx4Cre system

Multi-fluorescence reporter mouse strain: Rosa26rbw (Rainbow)

No recombination: EGFP
1. Recombination at : CFP
2. Recombination at : OFP

3. Recombination at : RFP

chicken beta actin promoter with a CMV enhancer

lox2272, loxN and loxP
ORF of fluorescent protein with beta globin poly A

UsinganendogenousreportertotraceDdx4positivecellsinvitro.

Rosa26rbw/+; Ddx4-Cre

Multi-fluorescent germ cells in the gonads of postnatal

Rosa26rbw/+;Ddx4-Cre female and male mice.

A B

50 m 50 m

PD28Rosa26rbw/+;Ddx4Creovary PD28Rosa26rbw/+;Ddx4Cretestis

HuaZhangetal.,PNAS,2012

Page 80 of 124
The Ddx4-positive cells from postnatal mouse ovaries are mitotically inactive

HuaZhangetal.,PNAS,2012

No Ddx4-expressing cells (0/1571) from postnatal mouse ovaries

proliferated during 72 h in vitro culture.

Ddx4expressingcellsfromPD8testes Ddx4expressingcellsfromPD8ovaries
Controlgroup Experimentalgroup

HuaZhangetal.,PNAS,2012

NoDdx4positivecolonyformedinlongtermculturedovariancells

HuaZhangetal.,PNAS,2012

Page 81 of 124
 TheDdx4negativeovarianclonalcellsfromRosa26rbw/+;Ddx4Cre
femalescannotdifferentiateintooocytesorgranulosacellsinvivo.

TheDdx4negativeovarianclonalcellsinculturearenotgermlineprogenitors

HuaZhangetal.,PNAS,2012

Conclusion:

Ddx4proteindoesnotcontainanytransmembrane
domain.UsingthesameDdx4antibody,isolatedcells
fromTillysandWuslabsturnedouttobedistinct.

Thereisstillalackofevidencethatneooogenesis
occursintheadultovary.Moreresearchisneeded.

FutureProspects:

1. Doesanyfollicularrenewaloccurinadultovaryunder
physiologicalorpathologicalconditions?

2.Aretheisolatedovarianstemcellsfunctional?

Page 82 of 124
References

Johnson,J.,Canning,J.,Kaneko,T.,Pru,J.K.,andTilly,J.L.(2004).Germlinestemcellsandfollicular
renewalinthepostnatalmammalianovary.Nature428,145150.

Johnson,J.,Bagley,J.,SkaznikWikiel,M.,Lee,H.J.,Adams,G.B.,Niikura,Y.,Tschudy,K.S.,Tilly,J.C.,
Cortes,M.L.,Forkert,R.,etal.(2005).Oocytegenerationinadultmammalianovariesbyputative
germcellsinbonemarrowandperipheralblood.Cell122,303315.

White,Y.A.,Woods,D.C.,Takai,Y.,Ishihara,O.,Seki,H.,andTilly,J.L.(2012).Oocyteformationby
mitoticallyactivegermcellspurifiedfromovariesofreproductiveagewomen.NatMed.

Zou,K.,Yuan,Z.,Yang,Z.,Luo,H.,Sun,K.,Zhou,L.,Xiang,J.,Shi,L.,Yu,Q.,Zhang,Y.,etal.(2009).
Productionofoffspringfromagermlinestemcelllinederivedfromneonatalovaries.NatCellBiol
11,631636.

Eggan,K.,Jurga,S.,Gosden,R.,Min,I.M.,andWagers,A.J.(2006).Ovulatedoocytesinadultmice
derivefromnoncirculatinggermcells.Nature441,11091114.

BristolGould,S.K.,Kreeger,P.K.,Selkirk,C.G.,Kilen,S.M.,Mayo,K.E.,Shea,L.D.,andWoodruff,T.K.
(2006).Fateoftheinitialfolliclepool:empiricalandmathematicalevidencesupportingitssufficiency
foradultfertility.DevBiol298,149154.

Zhang,H.,Zheng,W.,Shen,Y.,Adhikari,D.,Ueno,H.,andLiu,K.(2012).Experimentalevidence
showingthatnomitoticallyactivefemalegermlineprogenitorsexistinpostnatalmouseovaries.Proc
NatlAcadSciUSA109,1258012585.

Page 83 of 124
 ESHRE Annual Meeting 2014
Pre-Congress Course 13
Munich, Germany

Spermatogonia stem cells and

future fertility
Ans van Pelt PhD
Center for reproductive medicine
Academic Medical Center
Amsterdam,The Netherlands
[email protected]

Disclosure

I have nothing to disclose

I have no commercial or financial relationships with manufacturers
of pharmaceuticals, laboratory supplies or medical devices

Learning objectives
Understand the function of spermatogonial stem cells
(SSCs) in the testis
Understand spermatogenesis
Understand the germ cell depletion upon cancer
treatment
Understand the biological evidence for a possible fertility
preservation using SSCs
Learn about the translation of results on SSC culture and
transplantation in animal studies to a future SSC based
fertility preservation in men

Page 84 of 124
 Testis

SSCs are among the spermatogonia on the basal membrane of the seminiferous epithelium

Sperm production

Spermatogonial stem cells (SSCs) form the basis of lifelong spermatogenesis with daily
sperm production of 50-100 106 sperm. This requires a perfect balance between self
renewal and differentiation to sperm.

Ad Ap B Spc Spt

Selfrenewal vs differentiation
PLZF-/- mouse

The balance has shifted to differentiation resulting in SSC depletion

Buaas et al., 2004

Page 85 of 124
 Selfrenewal vs differentiation
GDNF overexpressing mouse GDNF +/- mouse

Balance shifted to self renewal Balance shifted to differentiation

Meng et al., 2000

Selfrenewal vs differentiation
Id4-/- mouse

Balance has shifted to differentiation resulting in SSC depletion

Oatley et al., 2011

Regulation selfrenewal and differentiation

Leydig
cells
Sertoli
FGF2 Sertoli
cells CSF1 cells
FGFR2 +
+ +
FSH
BMP4
c-Ret +
SSC activin A
GDNF Bcl6b +
GFR1

ID4 +
Peritubular Committed
cells - c-Kit
? Plzf
spermatogonia

Page 86 of 124
Spermatogenesis
Apale Adark
50% 50%

B spermatogonia

Pre-Leptotene

Leptotene

Zygotene

Pachytene

Diplotene

1st meiotic division

Meiosis

Spermiogenesis

Page 87 of 124
Breakthrough I: mouse SSC transplantation

Brinster & Averbock, 1994 Brinster & Nagano, 1998

Germ cell associations in the mouse

seminiferous epithelium

IV

IV
III

III

All associated germ cells stained blue

Page 88 of 124
 Germ cell associations in human
seminiferous epithelium

Germ cell associations in human

seminiferous epithelium

Muciaccia et al., 2013

SSC transplantation in various species

Autotransplantation:
Mouse to mouse
Bull to bull (Izadyar et al., Reproduction 2003)
Goat to goat (Honaramooz et al., Mol Reprod Dev 2003)
Rat to rat (Hamra et al., PNAS 2005)
Ram to ram (Ridriguez-sosa et al., Theriogenology 2006)
Dog to dog (Kim et al., Reproduction 2008)
Monkey to monkey (Herman et al., Cell Stem cells 2012)

Xenotransplantation:
Rat to mouse (Cloutier et al., Nature 1996)
Hamster to mouse (Ogawa et al., Biol Reprod 1999)
Rabbit/dog to mouse (Dobrinski et al., Biol Reprod 1999)
Baboon to mouse (Nagano et al., Biol Reprod 2001)
Bull to mouse (Izadyar et al., Reproduction 2002
Human to mouse (Nagano et al., Fert Steril 2002)

Page 89 of 124
Breakthrough II: in vitro propagation of SSCs

Kanatsu-Shinohara et al., 2003

Spermatogenesis and offspring of transplanted

cultured SSCs

Kanatsu-Shinohara et al, 2003

The human situation

Spermatogonial stem cells and future fertility

Page 90 of 124
Clinical problem
Causes of male infertility
Hyperprolactinemia
Hypogonadotrophic hypogonadism
Bilateral cryptorchidism
Orchitis
Genetic causes
Numerical and structural chromosome abnormalities
Y-chromosome deletions
Previous chemo- or radiotherapy

Silber & Repping, 2002, Visser & Repping, 2010

Clinical problem: loss of germ cells

Causes of male infertility
Hyperprolactinemia
Hypogonadotrophic hypogonadism
Bilateral cryptorchidism
Orchitis
Genetic causes
Numerical and structural chromosome abnormalities
Y-chromosome deletions
Previous chemo- or radiotherapy

Fertility problems after irrradiation

Number of sperm in
ejaculate after
irradiation (human)

Page 91 of 124
 Recovery
Difficult to predict
May not occur at all
May occur several years later
May result in azoo- or oligozoospermia

Silber & Rodriguez-Rigau, 1981

SSCs are sensitive for chemotherapy

Meistrich 2009

SSCs are sensitive for irradiation

Howell & Shalet, 2005

Page 92 of 124
 Fertility preservation
Cryopreservatie of sperm before onset cancer treatment

For prepubertal boys with cancer there is no means to preserve fertility with sperm

Theoretical solution
Cryopreservation of SSCs for later propagation and autotransplantation

Brinster, 2007

Translation to human
Propagation of human SSC

Page 93 of 124
 Culture of adult human testicular cells

Establishment of human germ cell clusters in vitro

Sadri Ardekani et al., JAMA 302, 2127-2134, 2009

Expression spermatogonial markers

Sadri Ardekani et al., JAMA 302, 2127-2134, 2009

Xenotransplantation adult human SSCs

Nude mouse
38 mg/Kg Busulfan treatment
cultured cells 4-6 weeks before transplantation

Colonization?

Page 94 of 124
 Human SSCs migrated after xenotransplantation

Human
adult testis

2 hours
after transplantation

10 weeks
after transplantation

Sadri Ardekani et al., 2009

Xenotransplantation readout
Human Culture days passage Number Number Dilution Human SSCs
Sample number of injected of factor fold
cells(105) colonies increase
/105 cells
Testicular cells culture

UMC0001 63 4 1.3 0
URO0003 14 1 3.5 0.7
14 1 0.2 12.5

URO0005 14 1 2.7 0.9

42 3 0.3 0
URO0008 28 3 0.7 3.6

URO0012 21 1 0.6 0
URO0021 28 3 0.1 0
56 7 0.4 0

28 2 2.55 2
47 5 3.1 0.8 133 53

GSCs subculture

URO0005 91 6 2.5 0
URO0021 77 7 2 1.25

84
141
8
12
0.5
1.9
5
2.6
8,870
18,450

Successful propagation of adult human SSCs

Sadri Ardekani et al., 2009

Adult vs prepubertal testis

Do prepubertal testicular cells also support SSC propagation in vitro?

Adult prepubertal

Page 95 of 124
 Prepubertal human testicular cell culture

Sadri Ardekani et al., 2011

Long term culture of human spermatogonia

Expression of spermatogonial markers in cultured testicular cells

Sadri Ardekani et al., 2011

Xenotransplantation of prepubertal human SSCs

Successful prepubertal human SSCs migration after xenotransplantation

Sadri Ardekani et al., 2011

Page 96 of 124
 Xenotransplantation of prepubertal human SSCs

Patient I.D Culture Number Number Dilution Human SSCs

days of of factor fold
(passage injected colonies increase
number) cells (105) /105 cells
Testicular cells culture
6.5 year old 70(5) 2.4 1

98(9) 3.6 0

46 (4) 2 0
8 year old
63 (6) 5.1 0.5 1.2 9.6
74 (7) 1.9 4

Successful propagation of prepubertal human SSCs

Sadri Ardekani et al., 2011

Leukemic patients

How to avoid risk of reintroduction of leukemic cells

Relapse intratubular transplantation of 20

leukemic cells

Jahnukainen K et al. 2001

Page 97 of 124
 Cancer cell elimination by FACS

Dovey et al., 2013

Cancer cell contamination in culture

Sadri-Ardekani et al., 2014.

SSC autotransplantation: parents perspective

Page 98 of 124
 Retrospective opinion of the parents

Van den Berg et al., Hum Rep 2007

Childrens Hospital Netherlands
162 parents (median 7 years post-diagnosis)
62% would have stored testicular biopsy

Sadri-Ardekani, et al., Fert Steril 2013

Childhood Cancer Center Iran
299 parents (children <12 year) (1 month to 19 years post
diagnosis)
54% would have stored a testicular biopsy

Retrospective opinion of the parents

Decision depends on
Chance of infertility by chemotherapy
Chance that autotransplantation will be successful in the future

What parents decide based on risks or chances of succes

Risk infertility 80% 65% would want a biopsy

Risk infertility 20% 35% would want a biopsy

Chance of success 80% 65% would want a biopsy

Chance of success 20% 26% would want a biopsy

Sadri Ardekani, et al., 2013

Summary SSC research

Year Author Highlighted findings Species
1966 Clermont Initial histological description of Apale and Adark spermatogonia Human
Model for renewal and differentiation of spermatogonia and
1971 Huckins Rat
existence of spermatogonial stem cells (SSCs)
First successful transplantation of testis-derived cells from one
1994 Brinster & Averbock Mouse
mouse to another resulting in donor derived F1 progeny
In vitro maintenance of SSCs for 4 months on a somatic feeder
1998 Nagano et al. Mouse
layer
Xenotransplantation of primate testis cell suspensions from one
1999 Schlatt et al. Macaque
primate into the testes of another
First report on successful colonization of mouse testes after
2002 Nagano et al. Human
xenotransplanting human SSCs
Kanatsu-Shinohara Prolonged in vitro propagation of SSCs using GDNF, without
2003 Mouse
et al. immortalization of the cells in culture
Proof of successful cryopreservation of testicular biopsies without
2005 Keros et al. Human
decreasing structural integrity
Kanatsu-Shinohara Long-term propagation of SSCs under serum free and feeder free
2005 Mouse
et al. conditions
Long-term propagation of adult SSCs in vitro with retainment of
2009 Sadri-Ardekani et al. Human
functionality
Long-term propagation of prepubertal SSCs with retainment of
2011 Sadri-Ardekani et al. Human
functionality
Production of functional sperm by infertile prepubertal macaques
2012 Hermann et al. Macaque
after autotransplantation, capable of fertilizing oocytes

Struijk et al., 2012

Page 99 of 124
 Conclusions
Spermatogonia are extremely sensitive for killing by
chemotherapy and irradiation.
Cryopreservation of SSCs for later transplantation is
the only option for prepubertal boys with cancer to
preserve their fertility and parents are eager to
preserve a testis biopsy from their son.
SSCs (including those of human) can survive and
proliferate in long term culture without losing their stem
cell characteristics to migrate to their niche upon
transplantation.
ALL cells can be eliminated during culture of SSCs.

Acknowledgements
Center for Reproductive Medicine AMC Department of Urology AMC
Hooman Sadri-Ardekani Andreas Meissner
Bita NickKolgh Theo de Reijke
Canan Mizrak Jean de la Rosette
Robin Struijk
Callista Mulder Department of Pediatric Oncology AMC
Marianne van de Wetering
Saskia van Daalen
Henk van den Berg
Cindy Korver Huib Caron
Hermien Roepers
Suzanne Hovingh Avicenna Research Institute, ACECR, Iran
Dirk de Rooij Mohammad Akhondi
Fulco van der Veen
Sjoerd Repping

granted by:

References I
Brinster RL, Avarbock MR (1994) Germline transmission of donor haplotype following spermatogonial
transplantation. Proceedings of the National Academy of Sciences of the United States of America 91: 11303-
11307
Brinster RL, Nagano M (1998) Spermatogonial stem cell transplantation, cryopreservation and culture. Seminars in
cell & developmental biology 9: 401-409
Buaas FW, Kirsh AL, Sharma M, McLean DJ, Morris JL, Griswold MD, de Rooij DG, Braun RE (2004) Plzf is
required in adult male germ cells for stem cell self-renewal. Nat Genet 36: 647-652
Clouthier DE, Avarbock MR, Maika SD, Hammer RE, Brinster RL (1996) Rat spermatogenesis in mouse testis.
Nature 381: 418-421
Dobrinski I, Avarbock MR, Brinster RL (1999) Transplantation of germ cells from rabbits and dogs into mouse
testes. Biology of reproduction 61: 1331-1339
Dovey SL, Valli H, Hermann BP, Sukhwani M, Donohue J, Castro CA, Chu T, Sanfilippo JS, Orwig KE (2013)
Eliminating malignant contamination from therapeutic human spermatogonial stem cells. J Clin Invest 123: 1833-
1843
Ginsberg JP, Carlson CA, Lin K, Hobbie WL, Wigo E, Wu X, Brinster RL, Kolon TF (2010) An experimental protocol
for fertility preservation in prepubertal boys recently diagnosed with cancer: a report of acceptability and safety.
Hum Reprod 25: 37-41
Hamra FK, Chapman KM, Nguyen DM, Williams-Stephens AA, Hammer RE, Garbers DL (2005) Self renewal,
expansion, and transfection of rat spermatogonial stem cells in culture. Proceedings of the National Academy of
Sciences of the United States of America 102: 17430-17435
Hermann BP, Sukhwani M, Winkler F, Pascarella JN, Peters KA, Sheng Y, Valli H, Rodriguez M, Ezzelarab M,
Dargo G, Peterson K, Masterson K, Ramsey C, Ward T, Lienesch M, Volk A, Cooper DK, Thomson AW, Kiss JE,
Penedo MC, Schatten GP, Mitalipov S, Orwig KE (2012) Spermatogonial stem cell transplantation into rhesus
testes regenerates spermatogenesis producing functional sperm. Cell Stem Cell 11: 715-726
Honaramooz A, Behboodi E, Blash S, Megee SO, Dobrinski I (2003) Germ cell transplantation in goats. Mol
Reprod Dev 64: 422-428
Howell SJ, Shalet SM (2005) Spermatogenesis after cancer treatment: damage and recovery. Journal of the
National Cancer Institute Monographs: 12-17

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References II
Izadyar F, Den Ouden K, Stout TA, Stout J, Coret J, Lankveld DP, Spoormakers TJ, Colenbrander B, Oldenbroek JK,
Van der Ploeg KD, Woelders H, Kal HB, De Rooij DG (2003) Autologous and homologous transplantation of bovine
spermatogonial stem cells. Reproduction (Cambridge, England) 126: 765-774
Izadyar F, Spierenberg GT, Creemers LB, den Ouden K, de Rooij DG (2002) Isolation and purification of type A
spermatogonia from the bovine testis. Reproduction (Cambridge, England) 124: 85-94
Jahnukainen K, Hou M, Petersen C, Setchell B, Soder O (2001) Intratesticular transplantation of testicular cells from
leukemic rats causes transmission of leukemia. Cancer Res 61: 706-710
Kanatsu-Shinohara M, Ogonuki N, Inoue K, Miki H, Ogura A, Toyokuni S, Shinohara T (2003) Long-term proliferation
in culture and germline transmission of mouse male germline stem cells. Biology of reproduction 69: 612-616
Keros V, Rosenlund B, Hultenby K, Aghajanova L, Levkov L, Hovatta O (2005) Optimizing cryopreservation of
human testicular tissue: comparison of protocols with glycerol, propanediol and dimethylsulphoxide as
cryoprotectants. Hum Reprod 20: 1676-1687
Kim Y, Turner D, Nelson J, Dobrinski I, McEntee M, Travis AJ (2008) Production of donor-derived sperm after
spermatogonial stem cell transplantation in the dog. Reproduction (Cambridge, England) 136: 823-831
Meistrich ML (2009) Male gonadal toxicity. Pediatr Blood Cancer 53: 261-266
Meng X, Lindahl M, Hyvonen ME, Parvinen M, de Rooij DG, Hess MW, Raatikainen-Ahokas A, Sainio K, Rauvala H,
Lakso M, Pichel JG, Westphal H, Saarma M, Sariola H (2000) Regulation of cell fate decision of undifferentiated
spermatogonia by GDNF. Science 287: 1489-1493
Muciaccia B, Boitani C, Berloco BP, Nudo F, Spadetta G, Stefanini M, de Rooij DG, Vicini E (2013) Novel stage
classification of human spermatogenesis based on acrosome development. Biology of reproduction 89: 60
Nagano M, McCarrey JR, Brinster RL (2001) Primate spermatogonial stem cells colonize mouse testes. Biology of
reproduction 64: 1409-1416
Nagano M, Patrizio P, Brinster RL (2002) Long-term survival of human spermatogonial stem cells in mouse testes.
Fertil Steril 78: 1225-1233
Oatley MJ, Kaucher AV, Racicot KE, Oatley JM (2011) Inhibitor of DNA binding 4 is expressed selectively by single
spermatogonia in the male germline and regulates the self-renewal of spermatogonial stem cells in mice. Biology of
reproduction 85: 347-356

References III
Ogawa T, Dobrinski I, Avarbock MR, Brinster RL (1999) Xenogeneic spermatogenesis following transplantation of
hamster germ cells to mouse testes. Biology of reproduction 60: 515-521
Rodriguez-Sosa JR, Dobson H, Hahnel A (2006) Isolation and transplantation of spermatogonia in sheep.
Theriogenology 66: 2091-2103
Sadri-Ardekani H, Akhondi MA, van der Veen F, Repping S, van Pelt AM (2011) In vitro propagation of human
prepubertal spermatogonial stem cells. JAMA 305: 2416-2418
Sadri-Ardekani H, Akhondi MM, Vossough P, Maleki H, Sedighnejad S, Kamali K, Ghorbani B, van Wely M, van der
Veen F, Repping S (2013) Parental attitudes toward fertility preservation in boys with cancer: context of different risk
levels of infertility and success rates of fertility restoration. Fertil Steril 99: 796-802
Sadri-Ardekani H, Homburg CH, van Capel TM, van den Berg H, van der Veen F, van der Schoot CE, van Pelt AM,
Repping S (2014) Eliminating acute lymphoblastic leukemia cells from human testicular cell cultures: a pilot study.
Fertil Steril, doi: 10.1016/j.fertnstert.2014.01.014
Sadri-Ardekani H, Mizrak SC, van Daalen SK, Korver CM, Roepers-Gajadien HL, Koruji M, Hovingh S, de Reijke
TM, de la Rosette JJ, van der Veen F, de Rooij DG, Repping S, van Pelt AM (2009) Propagation of human
spermatogonial stem cells in vitro. JAMA 302: 2127-2134
Silber SJ, Repping S (2002) Transmission of male infertility to future generations: lessons from the Y chromosome.
Human reproduction update 8: 217-229
Silber SJ, Rodriguez-Rigau LJ (1981) Quantitative analysis of testicle biopsy: determination of partial obstruction
and prediction of sperm count after surgery for obstruction. Fertil Steril 36: 480-485
Struijk RB, Mulder CL, van der Veen F, van Pelt AM, Repping S (2013) Restoring fertility in sterile childhood cancer
survivors by autotransplanting spermatogonial stem cells: are we there yet? BioMed research international 2013:
903142
van den Berg H, Repping S, van der Veen F (2007) Parental desire and acceptability of spermatogonial stem cell
cryopreservation in boys with cancer. Hum Reprod 22: 594-597
Visser L, Repping S (2010) Unravelling the genetics of spermatogenic failure. Reproduction (Cambridge, England)
139: 303-307

Page 101 of 124

ESHREAnnualMeeting2014,MunichJune29July2,2014
PreCongressCourse13
Ofstemcellsandgametes:moresimilaritiesthandifferences

Stemcellbasedapproaches
torestorespermatogenesis
inmonkeys
StefanSchlatt
CentreofReproductiveMedicineand Andrology
UniversityMnster,Germany

Disclosure
Nothingtodisclose
Ihavenocommercialorfinancialinterestswithmanufacturersof
pharmaceuticalorlaboratorysupplies/medicaldevices

LearningObjectives

Learningabout primate specific features of

spermatogonial stem cells (SSC)
Recognize the central role of SSCsfor malefertility
Distinguish the potentials and risks of currently
available and novel experimentalstrategies for male
fertility protection
Learn about perspectives of potentialfuture cell
based strategies for malegerm cell development in
vitro
Provide clinical perspectives for establishing
multidisciplinary programs onfertility preservation in
boys

Page 102 of 124

Observationofspermatogoniainprimates

Histological
Section

Whole
Mount

EhmckeJ,SchlattS.Identificationandcharacterizationofspermatogonial
subtypesandtheirexpansioninwholemountsandtissuesectionsfrom
primatetestes.MethodsMolBiol450:109118(2008)

WorkingHypothesis
Theextentofspermatogonialexpansiondetermines
thearrangementofspermatogenicstages

Onemitoticdivision:Smallclones Fourmitoticdivisions:Largeclones
Man Macaque
Mixedstages Longitudinalstages

HumanTestis

Arrangementof III
spermatogenic
stagesinprimate
species IV III IV II

Cynomolgusmonkey Marmoset

IV
VI
IV

Page 103 of 124

Confocalmicroscopy:clonalarrangementofspermatogonia

Immunohistochemistry:BrdU(green);acrosin(red)

EhmckeJ,LuetjensCM,SchlattS.Clonalorganizationofproliferatingspermatogonial
stemcellsinadultmalesoftwospeciesofnonhumanprimates,Macacamulattaand
Callithrixjacchus.BiolReprod72:293300(2005)

Clonalexpansionof
premeioticgermcells
inthehumantestis

Speciesspecificdifferences

Rodents
Veryhighmitoticexpansion(onestemcelldivisiongeneratesseveralthousandsperm)
Noselfrenewingprogenitorcellundersteadystateconditions,smallpoolofmitotically
activestemcells
Primates
Lowmitoticexpansion(onestemcelldivisiongeneratesfewsperm;human:16,monkey:256)
Selfrenewingprogenitorcellandstemcell

EhmckeJ,SchlattS.Arevisedmodelforspermatogonialexpansioninman:lessonsfromnonhumanprimates.
Reproduction132:673680(2006)

Page 104 of 124

 SchematicRepresentationofDefectsonHuman
GermCellDevelopmentAfterOncologicalTherapy

Spermatogonia Spermatocytes Spermatids

Differentiating Meiotic Postmeiotic

Stemcell Cells Cells Cells

Xray/chemo sensitive
DNA
damage?

quiescent proliferating

Recoveryoffertilityinboysaftercancertherapy
(DataprovidedbyKirsiJahnukainen,UniversityofHelsinki)

Testissize FSH

InhibinB FreeT

Effectsoftesticularirradiation
2yearspost10Gysingleirradiationinanimmaturerhesusmonkey

Complete or focal absence of germ cells

Page 105 of 124

 Testicular recovery after irradiation differs in prepubertal and
pubertal non-human primates
and can be enhanced by autologous germ cell transplantation.
Jahnukainen et al. Hum Reprod 26: 1945-1954 (2011)

Testes from prepubertal monkeys were more radiosensitive than pubertal monkeys

Spermin Biopsy
juveniletestes
andradiation
effectsin
prepubertal
rhesus
monkeys
singleexposure,
10Gy
Irradiation

3117 3083 3212

Preventionstrategy:
Exvivomaintenanceofstemcells
Cryopreservationofimmaturetesticulartissue

Multidisciplinaryteamofclinicians(oncologist,
paediatrician,urologist,andrologist,IVFgroup)
EthicsApproval(Experimentalprocedure)
Unilateralopentesticularbiopsies(severalincisions)
Dissectionintosmalltissuefragments(<1mm3)
Noenzymaticdigestion
CryopreservationusingDMSO(1.4M)asprotectant
SlowFreezingProtocol

Page 106 of 124

ESHRETaskForceforFertilityPreservationinSevereDiseases
MaleSubgroup:A European Perspective On Testicular Tissue Cryopreservation
For Fertility Preservation In Prepubertal And Adolescent Boys

MaleFertilityPreservation
EuropeanResearchNetworksandClinicalServiceActivities

EuropeanAcademicMedicalCentres
holdingcryobankedimmature
humantestesfromoncologicalpatients
FertiProtect(Germany)
ZUBrussels,Belgium
PROSPERMAinCECOS CatholicUniv,Brussels,Belgium
(Centred`EtudeetdeConservationdes RigshospitaletKopenhagen,Denmark
OeufsetduSpermehumains) HopitauxdeRouen,France
GRECOT CeRA,Germany
(Groupderecherchesurlaconservation AMC,Amsterdam,Netherlands
KarolinskaUniversityHospital,Sweden
dutissueovarienettesticulaire
UniversityofEdinburgh,UK
(France)
NordicNetworkforFertilityPreservationinChildren
(Norway,Sweden,Denmark)

Howtogeneratespermfrom
thecryopreservedtissue?

- Germ cell transplantation

- Testicular Grafting
- In vitro spermatogenesis

Page 107 of 124

 DevelopmentofGermCellTransplantation

1994 Spermatogenesis followingmalegermcelltransplantation.

(BrinsterandZimmermann,PNAS91:11298)
1996 Ratspermatogenesis inmousetestis.
(Clouthieretal.,Nature381:418)
1996 Reconstitutionofspermatogenesisfrom frozenspermatogonial
stemcells.(Avarbocketal.,NatMed2:693)
1998 Culture ofmousespermatogonialstemcells.
(Naganoetal.,Tissue&Cell30:389)
1999 Germcelltransferintorat,bovine,monkeyandhumantestes.
(Schlattetal.,HumReprod14:144)
2001 Primatespermatogonialstemcellscolonizemousetestes.
(Naganoetal.,BiolReprod 64:1409)
2001 Transgenicmiceproducedbyretroviraltransductionofmale
germlinestemcells. (Naganoetal.PNAS98:13090)
2002 GermcelltransplantationintoXirradiatedmonkeytestes.
(Schlattetal.HumReprod(17:55)

Xenologoustransplantationofprimatespermatogonia

Baboongermcellcolonisingmousetestes

Naganoetal.,2001Primatespermatogonial
stemcellscolonisemousetestes.
BiolReprod64:14091416 Baboongermcellcolonisingmousetestes
aftercryopreservation

Naganoetal.,2002Longtermsurvival
ofhumanspermatogonialstemcells Colonisationofprimate
inmousetestes. spermatogoniainmousetestes
FertilSteril78:122533

GermCellInfusionintothePrimateTestis

Microinjectionofseminiferous
tubules:
difficultandinefficient

Injectionsinto
efferentducts:
surgicallydemanding
andinefficient

Injectionsintothe
retetestis:
Easy,efficientand
reproducible

Involutedrecipienttestis Ultrasound
guidance

Page 108 of 124

 Germ Cell Transfer into Rat, Bovine, Monkey and
Human Testes Schlatt et al., Hum Reprod 14: 144-150 (1999)

1 2 3

4 5 6

TestisVolumeFollowingIrradiationandGermCellTransfer
SchlattS,FoppianiL,RolfC,WeinbauerGF,NieschlagE.Germcelltransplantationinto
Xirradiatedmonkeytestes. HumReprod17:5562(2002)
Testisvolume(ml)

Right testis Left Testis

Germ cell transfer (35 wks) Saline Injection

6105 6105

6102 6102

Page 109 of 124

 Testicularrecoveryafter
irradiationdiffersin
prepubertalandpubertal
nonhumanprimates,and
canbeenhancedby
autologousgermcell
transplantation.
Jahnukainenetal.Hum
Reprod26:19451954
(2011)

Spermatogonialstemcelltransplantationinto
Rhesustestesregeneratesspermatogenesis
producingfunctionalsperm.
Hermannetal.CellStemCell11:715726(2012)

Reproductiontroubled
bydarkness

Page 110 of 124

 TesticularGrafting

Transplantationofthe
stemcellanditsniche

DwarfMales

Spermfromneonatalmammaliantestesgraftedinmice
Honaramoozetal.,Nature2002

Schlattetal., Progenyfromspermobtained
afterectopicgraftingofneonatalmouse
testes.BiolReprod68:23312335(2003)

Page 111 of 124

 Endocrine
response of
castrated nude
mice after
grafting:

Substitution of
androgens
and suppression
of FSH.

Neonatal pig testis Pig graft at week 12

Sperm morphology and oocyte-
activating competence of sperm
from testicular grafts
Mouse sperm Mouse pronuclei

Pig grafts at week 18 Cryopreserved pig graft

Pig sperm Pig pronuclei

Neonatal goat testis Goat graft at week 10

Goat sperm Goat pronuclei

Histology of grafts from pig

and goat testis (All oocytes are from mice)

Limited survival of adult human testicular tissue as ectopic xenograft

Schlatt et al. Hum Reprod 21: 384-389 (2005)

See also: Geens et al. Spermatogonial survival after grafting human testicular tissue
to immunodeficient mice. Hum Reprod 21: 389-395 (2005)

Page 112 of 124

Completespermatogenesisin
orthotopicbutnotinectopic
transplantsofautologously
graftedmarmosettesticular
tissueLuetjensetal.Endocrinology149:
17361747(2008)

Testicularxenografts:Anovelapproachtostudy
cytotoxicdamageinjuvenileprimatetestis
Jahnukainenetal.,CancerResearch66:38133818(2006)
Pretreatment Busulfan

Control

Conclusion:Xenograftedmonkeytestistissue
showsthesamechangestotoxinsasintact
testes.

Irradiationcausesacuteandlongterm
Organculture(24hours)

spermatogonialdepletioninculturedand
xenotransplantedtesticulartissuefrom
juvenilenonhumanprimatesJahnukainenet
al.,Endocrinology2007;148:55415548
Contro 4 Gy
l

Contro 4 Gy
l
Xenografting(4months)

Contro 0.5 Gy
l

1 Gy 4 Gy

Page 113 of 124

Effectofcoldstorageand
cryopreservationof
immaturenonhuman
primatetesticulartissueon
spermatogonialstemcell
potentialinxenografts.
Fresh 24hrsonice
Jahnukainenetal.,Hum
Reprod.2007;22:10607

On ice

Fridge -20C

Liquid nitrogen

0.7MDMSO 1.4MDMSO

Survival Graft weight Grafts with A-spg Grafts with B-spg Grafts with spct
Seed 0.7M Seed 1.4M
grafted/ mg or sptd
recovered

Ctrl 51% 33/64 5.40.5 100% 79% 12%

Fresh 52% 14/27 7.31.2 100% 100% 36%

DMSO 26% 6/23 4.50.9 83% 67% 0%

EG 7% 2/27 6.51.5 100% 100% 0%

Without 0% 0/23 - - - -

Fertilitypreservationaftersterilizingtherapythrough
autologousectopicgraftingofcryopreserved
testiculartissueinprepubertalmonkeys
Jahnukainenetal.,CancerResearch72:517478(2012)

Autograftingintoscrotalarea:spermproduction(manygrafts!)
Xenograftingorectopicgrafting:nofullspermatogenesis

Page 114 of 124

 CentreofReproductive
Acknowledgements MedicineandAndrology,
UniversityMnster,
Germany
CentreforResearchin
ReproductivePhysiology, JensEhmcke BirgitWesternstrer
UniversityofPittsburgh JrgGromoll JanBerndStukenborg
SchoolofMedicine,PA, SabineKliesch JoachimWistuba
USA jfie MichaelZitzmann EberhardNieschlag

KirsiJahnukainen
BhavikaJoshi
TonyPlant
SureshRamaswamy
DavidSimorangkir
ScottHergenrother
KathrinGassei

GIF
GermanIsraeliFoundation
forScientificResearchand
Development

Page 115 of 124

 UPCOMING ESHRE EVENTS
// ESHRE CAMPUS EVENTS

ESHREs 30th Annual Meeting Epigenetics in reproduction

www.eshre.eu/lisbon
www.eshre2014.eu

Munich, Germany Lisbon, Portugal

29 June - 2 July 2014 26-27 September 2014

Endoscopy in reproductive medicine Making OHSS a complication of the past:

www.eshre.eu/endoscopyoct State-of-the-art use of GnRH agonist
triggering www.eshre.eu/thessaloniki

Leuven, Belgium Thessaloniki, Greece

15-17 October 2014 31 October-1 November 2014

From gametes to blastocysts Controversies in endometriosis and

a continuous dialogue adenomyosis
www.eshre.eu/dundee www.eshre.eu/liege

Dundee, United Kingdom Lige, Belgium

7-8 November 2014 4-6 December 2014

Bringing evidence based early pregnancy An update on preimplantation genetic

care to your clinic screening (PGS)
www.eshre.eu/copenhagen www.eshre.eu/rome

Copenhagen, Denmark Rome, Italy

11-12 December 2014 12-13 March 2014

For information and registration: www.eshre.eu/calendar

or contact us at [email protected]

Page 116 of 124

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