long-term oocyte-like cell development in cultures derived … · 2015-11-11 · long-term...

Research ArticleLong-Term Oocyte-Like Cell Development inCultures Derived from Neonatal Marmoset Monkey Ovary

Bentolhoda Fereydouni1 Gabriela Salinas-Riester2 Michael Heistermann3

Ralf Dressel4 Lucia Lewerich1 Charis Drummer1 and Ruumldiger Behr1

1Stem Cell Biology Unit German Primate Center-Leibniz Institute for Primate Research Kellnerweg 4 37077 Gottingen Germany2Microarray and Deep-Sequencing Core Facility University Medical Center Gottingen (UMG) Justus-von-Liebig-Weg 1137077 Gottingen Germany3Endocrinology Laboratory German Primate Center-Leibniz Institute for Primate Research Kellnerweg 4 37077 Gottingen Germany4Department of Cellular and Molecular Immunology University of Gottingen Humboldtallee 34 37073 Gottingen Germany

Correspondence should be addressed to Rudiger Behr rbehrdpzeu

Received 28 May 2015 Revised 28 July 2015 Accepted 28 July 2015

Academic Editor Irma Virant-Klun

Copyright copy 2016 Bentolhoda Fereydouni et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

We use the common marmoset monkey (Callithrix jacchus) as a preclinical nonhuman primate model to study reproductive andstem cell biology The neonatal marmoset monkey ovary contains numerous primitive premeiotic germ cells (oogonia) expressingpluripotent stem cell markers including OCT4A (POU5F1) This is a peculiarity compared to neonatal human and rodent ovariesHere we aimed at culturingmarmoset oogonia fromneonatal ovariesWe established a culture system being stable formore than 20passages and 5 months Importantly comparative transcriptome analysis of the cultured cells with neonatal ovary embryonic stemcells and fibroblasts revealed a lack of germ cell and pluripotency genes indicating the complete loss of oogonia upon initiation ofthe culture From passage 4 onwards however the cultured cells produced large spherical free-floating cells resembling oocyte-likecells (OLCs) OLCs strongly expressed several germ cell genes and may derive from the ovarian surface epithelium In summaryour novel primate ovarian cell culture initially lacked detectable germ cells but then produced OLCs over a long period of timeThis culture system may allow a deeper analysis of early phases of female primate germ cell development andmdashafter significantrefinementmdashpossibly also the production of monkey oocytes

1 Introduction

It is a long-held opinion in reproductive biology that infemales of most species including the human the postnatalgerm cell pool is limited in number and cannot be replen-ished or even expanded Early studies in the rat showedthat oogonia the proliferating female germ line progenitorcells that enter meiosis to produce oocytes were foundonly during fetal development [1] Human females are alsothought to be born with a nonrenewable pool of germ cellfollicles which declines with age [2] In postnatal humanovaries oogonia were found only very sporadically and wereundetectable by the age of two years [3] A lack of evidencefor postnatal germ cell proliferation in human ovaries wasalso reported by Liu et al [4] Instead by far most of

the proliferation-competent oogonia in the human ovaryentered meiosis by the end of the second trimester ofgestation [5 6] Recent studies using transgenic mice andnonhuman primates also reported a lack of evidence forgerm cell proliferation in adult mouse [7 8] and monkey [9]ovaries However these data supporting the view of a fixedpostnatal female germ cell pool are profoundly challengedby reports starting a decade ago [10 11] suggesting replen-ishment of the ovarian germ cell pool in mice The existenceof mitotically active germ line stem cells in postnatal mouseand human ovaries was suggested by the isolation molecularcharacterization and transplantation of cell populations [1012] Probably the strongest evidence so far for a germ line stemcell pool in the ovary was provided by Zou et al [13] whogenerated mouse female germ line stem cells (FGSCs) with

Hindawi Publishing CorporationStem Cells InternationalVolume 2016 Article ID 2480298 17 pageshttpdxdoiorg10115520162480298

2 Stem Cells International

the ability to reconstitute oogenesis and produce offspringafter transplantation Other authors reported the productionof oocyte-like cells in vitro from cultures of adult ovarian sur-face epithelium (OSE) [14ndash16] In summary currently thereare several apparently contradictory reports which providedata supporting or negating generation of new oocytes in thepostnatal rodent and primatemdashincluding the humanmdashovaryAdding to this complex situation Dyce et al [17] reportedthat even fetal porcine skin cells can form oocyte-like cells(OLCs) in vitro

We use the common marmoset monkey (Callithrix jac-chus) as a nonhuman primate model to study reproductiveand stem cell biology In contrast to other species usedin reproductive biology the marmoset monkey still hasnumerous oogonia in the neonatal ovary which robustlyexpress the pluripotency associated markers OCT4A LIN28and SALL4 the germ cell marker VASA and the proliferationmarker KI-67 [18] Therefore these oogonia in the neonatalmarmoset ovary share important pluripotency markers withmarmoset monkey embryonic stem (ES) cells [19] We failedto detect this proliferating and pluripotency-marker-positivecell population in the one-year-old and adult ovary [18]indicating a fast postnatal oogonial clearing also in thisnonhuman primate species Here we report studies onthe culture of neonatal marmoset ovarian cells We origi-nally aimed at culturing the proliferating marmoset monkeyoogonia exhibiting a pluripotency signature similar to mar-moset monkey embryonic stem cells Hence we establisheda mouse embryonic fibroblast- (MEF-) based cell culturesystem which allowed the long-term culture of ovarian cellsHowever germ cell and pluripotency marker expression waslost in the first passages indicating the complete loss of theoogonia After a few passages however germ cell markerexpression recovered and we observed in later passages thedevelopment of large spherical free-floating cells which wecalled oocyte-like cells (OLCs) due to their morphologicalresemblance with the cells reported by Dyce et al [17] OLCshad a diameter up to sim40 120583m and strongly expressed severalgerm cell markers This study demonstrates the developmentof oocyte-like cells in long-term ovarian cell cultures froma translational and experimentally accessible nonhumanprimate species

2 Materials and Methods

21 Animals Marmoset monkeys (Callithrix jacchus) for thisstudy were obtained from the self-sustaining breeding colonyof the German Primate Center (Deutsches Primatenzen-trum DPZ) The German Primate Center is registered andauthorised by the local and regional veterinary governmentalauthorities (Reference number 1229103311900 PK LandkreisGottingen) Health and well-being of the animals werecontrolled daily by experienced veterinarians and animalcare attendants The legal guidelines for the use of animalsand the institutional guidelines of the DPZ for the careand use of marmoset monkeys were strictly followed Theanimals were pair-housed in a temperature- (25 plusmn 1∘C)and humidity-controlled (65 plusmn 5) facility Illumination wasprovided by daylight and additional artificial lighting on

a 1200 1200-hour light dark cycleThe animals were fed adlibitum with a pelleted marmoset diet (ssniff SpezialdiatenSoest Germany) In addition 20 g mash per animal wasserved in the morning and 30 g cut fruits or vegetablesmixed with noodles or rice was supplied in the afternoonFurthermore once per week mealworms or locusts wereserved in order to provide adequate nutrition Drinkingwaterwas always available

In captivitymarmosets sometimes give birth to triplets oreven quadruplets However the mother is usually able to feedand rear only two neonates which is the normal litter size offree-living marmosets Therefore the neonates from tripletbirths were used to collect organs for this study Marmosetmonkey ovaries were obtained from six neonatal animals(postnatal days 1ndash5) All animals were narcotized with Pento-barbital (Narcoren 005mL intramuscular) and euthanizedby an experienced veterinarian with an intracardial injectionof 05mL Pentobarbital before a lack of nourishment causedsuffering of the animals Wherever applicable the ARRIVEguidelines were followed

22 Numbers of Animals Altogether ovaries from 6 neonatalmarmosets were used in this study 5 pairs for culture and onepair as reference for transcriptome analysis

23 Neonatal Common Marmoset Monkey Ovarian Cell Cul-tures Neonatal ovaries (approximate dimensions 2mm times1mm times 1mm see also [18]) were first collected and washedin a Petri dish containing cold DPBS Fat and blood cellswere removed and the whole ovary including the OSE wastransferred to fresh buffer and minced with sterile scissorsMinced ovaries were then transferred to a 15mL falcon tubeAfter gentle centrifugation DPBS was removed and ovariantissue fragments were resuspended in DMEMF12 mediumcontaining collagenase (Sigma C2674) and DNase and keptfor 45 minutes at 37∘C Every 5 to 10min the ovaries weregently pipetted to disintegrate the tissue Finally 10 FBSwas added to inactivate the enzyme In order to removelarger undigested tissue fragments the cells were passedthrough 70 120583m strainer (BD USA) and were centrifuged at200 g for 10min Then the supernatant was removed and thecells were resuspended in culture medium and transferredon a prepared feeder layer of mouse embryonic fibroblast(MEF) feeder at a concentration 3ndash5 times 105 cells per 5 cmwell After one week large colonies were individually pickedwith a microprobe (FST Heidelberg Germany 10032-13)and digested with Accutase (Life Technologies Germany) for4minThen the cells were centrifuged at 300 g for 10 minutesand resuspended in the culture medium for further passagesCultures were maintained at 37∘C under 5 CO

2and 5

oxygen in a humidified incubator

24 Culturing Neonatal Common Marmoset Ovaries Differ-ent cell culture conditions were tested Finally this conditionwas selected DMEMF12 supplemented with FBS (10)PenStrep amphotericin B and human LIF (10 120583gmL) onan irradiated mouse embryonic fibroblast (MEF) feeder celllayer For further passages (usually after 7ndash10 days) colonies

Stem Cells International 3

were mechanically removed with a sterile probe digestedwith Accutase and placed on a newly prepared MEF layer

25 CulturingMarmosetMonkey Embryonic StemCells Mar-moset monkey ES cells were basically cultured as describedpreviously [20] Only for passaging of the ES cells StemProAccutase (Life Technologies) was used instead of trypsin-EDTA with subsequent mechanical dissociation

26 Immunofluorescence Staining For immunofluorescencestainingmediumwas removed and cell colonieswerewashedtwice in PBS and then fixed in 4 PFA for 15 minutesCells were permeabilized by 01 Triton X-100 in PBS for10 minutes at room temperature Primary antibody (VASARampD Systems AF2030) was diluted in 3 BSA-PBS andcolonies were incubated for 1 h at 37∘C Cells were washedtwice in PBS The secondary antibody which was dilutedin 3 BSA-PBS was added to the cells and incubated for20 minutes in a dark box at 37∘C Cells were washed againtwice in PBS and 5 DAPI-PBS was added Cells werewashed again with PBS and mounted with CitiFluor (ScienceServices AF1 GlycerolPBS solution) For negative controlsthe primary antibody was (i) omitted or (ii) replaced byIgG isotopes DAPI staining of the OLCs was performedin 5 DAPI-PBS for 5 minutes followed by two washes inPBS

27 Histology and Immunohistochemistry Staining Histologyof the colonies was performed after mechanically detachingof whole MEF layer including the ovarian cell coloniesfrom the cell culture well After detachment the randomlyarranged MEF layer was fixed in Bouinrsquos solution for 3hours washed several times for at least 24 h in 70 EtOHand then embedded in paraffin wax The resulting cellculture conglomerate was randomly sectioned Due to thelarge number of colonies sufficient sections of ovarian cellcolonies in different orientations were available for histo-logical analysis Immunohistochemistry was performed asdescribed recently [18] using the following primary anti-bodies OCT4A (2890S Cell Signaling Technology Ger-many 1 100) LIN28A (3978S Cell Signaling Technol-ogy 1 100ndash1 200) SALL4 (ab57577 Abcam UK 1 200)and VASA (DDX4 AF2030 RampD Systems Germany1 100)

28 Western Blot Analysis Western blotting was performedaccording to standard procedures In brief 20mg of frozentissue per samplewas used Proteinswere isolated bymechan-ical destruction of the tissue in a TissueLyser at 50HZ(Qiagen Hilden Germany) The samples were denatured for5 minutes at 95∘C Samples were then run on a 10 SDS-page gel in Tris HCl buffer pH 88 and then semi-dry-blotted onto a PVDFmembrane (150mA for 1 h) Blocking ofunspecific bindingwas achieved by incubating themembranein 5 skim milk powder diluted in TBS for 1 h Primaryantibodies against VASA (AF2030 RampD Systems 1 2000)and 120573-actin (Santa Cruz SC-1616-R 1 5000) were dilutedin blocking buffer Membranes were incubated in primary

antibody solutions for 16 hours at 4∘C and then washedthree times with blocking solution supplemented withTween 20 After incubation with the horseradish peroxidase-coupled secondary antibody (1 h at 20∘C) the membranewas washed again Detection of bound antibody was per-formed using the Amersham ECL Western Blotting Detec-tion Reagents Kit (RPN2106) Signals were detected and doc-umented using the ChemoCam Imager (INTAS GottingenGermany)

29 Transcriptome Analysis For transcriptome analysis twoneonatal marmoset ovaries colonies from P4 from twodifferent individual animals (100ndash300 colonies per sample)marmoset skin fibroblasts and marmoset ES cells wereanalyzed Primary fibroblasts were obtained and cultured asdescribed recently [21] RNA was isolated using the TRIzolReagent (Life Technologies) according to the manufacturerrsquosinstructions RNAquality was assessed bymeasuring the RIN(RNA Integrity Number) using an Agilent 2100 Bioanalyzer(Agilent Technologies Palo Alto CA) Library preparationfor RNA-Seq was performed by using the TruSeq RNASample Preparation Kit (Illumina Cat number RS-122-2002)starting from 500 ng of total RNA Accurate quantitationof cDNA libraries was performed by using the QuantiFluordsDNA System (Promega) The size range of final cDNAlibraries was determined applying the DNA 1000 chip on theBioanalyzer 2100 from Agilent (280 bp) cDNA libraries wereamplified and sequenced by using the cBot and HiSeq2000from Illumina (SR 1 times 50 bp 5-6GB ca 30ndash35 millionreads per sample) Sequence images were transformed withIllumina software BaseCaller to bcl files which were demul-tiplexed to fastq files with CASAVA v182 Quality checkwas done via fastqc (v 0100 Babraham Bioinformatics)The alignment was performed using Bowtie2 v210 to thecDNA for Callithrix jacchus Data were converted and sortedby samtools 0119 and reads per gene were counted viahtseq version 054p3 Data analysis was performed byusing RBioconductor (302212) loadingDESeq gplots andgoseq packages Candidate genes were filtered to a minimumof 4x fold change and FDR-corrected 119901 value lt 005The datadiscussed in this paperwere generated in compliancewith theMIAME guidelines and have been deposited in NCBIrsquos GeneExpression Omnibus and are accessible through GEO Seriesaccession number GSE 64966

210 Quantitative Reverse Transcriptase Polymerase ChainReaction (RT-qPCR) Analysis RT-qPCR was carried outas described previously [18] Total RNA was extractedfrom different passages of colony-forming cells Around50ndash100 colonies per passage were pooled and analyzedThree independent cultures (derived from three differentanimals) were analyzed Each RNA sample was analyzedin triplicate As positive controls neonatal marmoset ovaryand marmoset embryonic stem cells were used Marmosetmonkey fibroblasts served as a biological negative controlfor pluripotency and germ cell markers Primers are listed inSupplementary Table 1 in Supplementary Material availableonline at httpdxdoiorg10115520162480298 For oocyte-like cell RNA extractions altogether 28 cells from different


Table 1 Primer sequences sizes of amplicons and concentration of respective primers

Primer Primer sequence PCR product size (bp) Concentration [nM]Cj GAPDH Fw 51015840-TGCTGGCGCTGAGTATGTG-31015840 64 300Cj GAPDH Re 51015840-AGCCCCAGCCTTCTCCAT-31015840 50Cj LIN28 Fw 51015840-GACGTCTTTGTGCACCAGAGTAA-31015840 67 300Cj LIN28 Re 51015840-CGGCCTCACCTTCCTTCAA-31015840 50Cj SALL4 Fw 51015840-AAGGCAACTTGAAGGTTCACTACA-31015840 77 900Cj SALL4 Re 51015840-GATGGCCAGCTTCCTTCCA-31015840 50Cj VASA Fw 51015840-TGGACATGATGCACCACCAGCA-31015840 210 50Cj VASA Re 51015840-TGGGCCAAAATTGGCAGGAGAAA-31015840 900Cj OCT4A Fw 51015840-GGAACAAAACACGGAGGAGTC-31015840 234 300Cj OCT4A Re 51015840-CAGGGTGATCCTCTTCTGCTTC-31015840 50Cj PRDM1 Fw 51015840-ATGAAGTTGCCTCCCAGCAA-31015840 147 50Cj PRDM1 Re 51015840-TTCCTACAGGCACCCTGACT-31015840 50Cj PRDM14 Fw 51015840-CGGGGAGAAGCCCTTCAAAT-31015840 91 50Cj PRDM14 Re 51015840-CTCCTTGTGTGAACGTCGGA-31015840 50Cj DAZL Fw 51015840-GAAGAAGTCGGGCAGTGCTT-31015840 70 50Cj DAZL Re 51015840-AACGAGCAACTTCCCATGAA-31015840 50Cj DPPA3 Fw 51015840-GCGGATGGGATCCTTCTGAG-31015840 129 50Cj DPPA3 Re 51015840-GAGTAGCTTTCTCGGTCTGCT-31015840 50Cj NOBOX Fw 51015840-GAAGACCACTATCCTGACAGTG-31015840 320 50Cj NOBOX Re 51015840-TCAGAAGTCAGCAGCATGGGG-31015840 50Cj SCP3 Fw 51015840-TGGAAAACACAACAAGATCA-31015840 60 50Cj SCP3 Re 51015840-GCTATCTCTTGCTGCTGAGT-31015840 50

passages were collected and randomly divided into twogroups The RNA was isolated with the RNeasy MICRO kit(Qiagen) according to the manufacturerrsquos instructions Toanalyze the relative gene expression level changes duringthe culture within one passage colonies from P7 wereseeded into 8 separate wells The cells from two wells wereharvested for analysis at days 2 4 6 and 8 Primer sequencessizes of PCR products and primer concentrations are givenin Table 1

211 Hormone Measurements Measurement of progesteronein the selected cell culture medium samples after at least3 days of conditioning was performed using an enzymeimmunoassay (EIA) using antiserum raised in sheep againstprogesterone-11-hemisuccinate-BSA as described by Heister-mann and colleagues [22] Freshmediumwas used as controlEstradiol-17120573 was determined using an EIA according toHeistermann et al [23] with the exception that 17120573-estradiol-6-horse-radish-peroxidase was used as label Both steroidmeasurements were performed in undiluted samples andfresh medium was used as control

212 Cell Transplantation Assay In order to test the cul-tured ovarian cells for their regenerative and tissue neomor-phogenesis potential we subcutaneously injected sim106 cellsper mouse The cells were obtained from Accutase-treatedovarian cell colonies from the 5th passage Four female adultRAG2minusminus120574cminusminus mice lacking B T and NK cells were usedThe technical procedure has been described previously forneonatal testis tissue [24]

3 Results

31 General Observations and Morphology of the PrimaryCell Cultures We established a long-term primary culture ofovarian cells This included passaging as well as expansionof the cells Five individual primary cultures of neonatalmarmoset ovaries were performed and run up to 6 monthswith very similar morphology and kinetics The maximumpassage number was 23 Then the cells stopped prolifera-tion Initially relatively small ovarian cell colonies (OCCs)formed which could be distinguished from the MEFs bythe morphology of the cells and the coloniesrsquo boundaries(Figure 1(a))TheOCCs quickly increased in size forming bigcolonies with diameters up to 1000 120583m (Figure 1(b)) within afew daysThe cells forming the OCCs exhibited an epitheloidphenotype as judged from morphology (Figure 1(d)) thatis they are apparently polar with apical nuclei and nointracellular matrix was visible between the cells forming thecolonies by lightmicroscopyHowever the cells lacked typicalepithelial markers such as E-cadherin (CDH1) For furtherdetails see below In higher passages the individual OCCsbecame smaller (compare Figures 1(b) and 1(c)) Some OCCsdeveloped in their centers a second layer of cells on top ofthe primary cell layer (Figure 1(c)) The morphology of thecolonies was faintly reminiscent of primate ES cell colonies[20] (Figure 1(e)) Colonies with the morphology of OCCswere never observed in pure mouse embryonic feeder cellcultures

32 OCCs Lack a Germ Cell Population in Early PassagesIn order to initially compare the neonatal ovary-derived cell


P1 early

(a)

P1 late

(b)

P10

(c)

(d)

200120583m

(e)Figure 1 Morphology of ovarian cell colonies (OCCs) (andashc)Morphology of cell colonies in the first passage and in higher passages (d) HampEstaining of cross sections of colonies Cells have an epitheloid morphology with rather apical nuclei (e) Morphology of a marmoset monkeyES cell colony

colonies on the transcriptome level with reference sampleswe performed a comprehensive transcriptome analysis ofOCCs by deep sequencing and compared the data set withthe transcriptomes of neonatal ovaries which served asstarting material and contained oogonia marmoset ES cellsas a reference for pluripotent cells and skin fibroblastswhich represent prototypic mesenchymal cells Each indi-vidual samplersquos transcriptome was represented by at least12Mio reads (Supplementary Figure 1) About 40000 dif-ferent transcripts were detected in each individual sample(Supplementary Figure 2) We used low passage numbersamples (passage 4) in order to obtain data from cells withoutextensive cell culture adaptation artefacts Due to the verylimited material only two independent OCC samples andtwo neonatal ovaries could be analyzedHowever already thissmall set of samples provided valuable insights (Figure 2)TheOCC samples were clearly distinct from native ovary andfibroblasts (Figure 2(a)) In contrast the differences betweenthe OCCsrsquo and the ES cellsrsquo transcriptomes were smallerImportantly the PCA plot (Figure 2(a)) indicates fundamen-tal differences between the transcriptomes of ovaries andOCCs Notably the top 50 differentially expressed genesbetween native ovaries and OCCs revealed two major facts(Figure 2(b)) (1) Almost all differentially expressed geneswere overrepresented in the native ovary This suggests thatthe OCCs generally represent a subpopulation of the wholecell population constituting the native ovary and that nocompletely different or novel cell type developed in cultureat least not in detectable quantities (2) Among the top50 differentially expressed genes are numerous germ-cell-specific genes likeMAEL RNF17 TEX12 TDRD9MOV10L1

NOBOX ZP3 FIGLA SOHLH2 DAZL and SYCP2 In factseveral germ cell genes including DAZL MAEL RNF17TEX12 TEX101 and TDRD were totally undetectable in thetranscriptomes of the OCCs Other transcripts like OCT4LIN28 and VASA were also extremely low or absent Thisstrongly indicates the complete loss of the typical neonatalovarian germ cell population including postmigratory PGCsoogonia and oocytes in the cell culture

The comparison between the OCCs and the fibroblastsrevealed an increased expression of many genes in the OCCs(Figure 2(c)) The upregulated genes include COL2A1 thekeratin gene KRT36 and VCAM1 Importantly however nogerm cell gene was found upregulated in OCCs comparedto fibroblasts further substantiating the absence of germcells from the OCC cultures The comparison of the top 50differentially expressed genes between the OCCs and the EScells showed thatmost genes were upregulated in ES cells likeTDGF1 LIN28A OCT4 (POU5F1) and NANOG Only a fewgenes including the dual specificity phosphatase 13 (DUSP13)and the serine peptidase inhibitor Kazal type 1 (SPINK1)were upregulated in OCCs (Figure 2(d))

The detailed cellular identity of the OCCs could not bedetermined so far Many of the cells of the OCCs were pro-liferation marker Ki-67-positive (Supplemental Figure S3)Although the cells had an epitheloid shape we failed todetect E-cadherin in the transcriptome data as well as byIHC (data not shown) Also cytokeratins as characteristicproteins of epithelial cells were only barely represented in thetranscriptomes Vimentin as typical protein of cells of mes-enchymal origin was expressed at medium levels (sim50 ofneonatal ovary levels and 30 of fibroblast levels) However


Component 1 (4270)

Com

pone

nt 2

(23

79

)

Neonatal ovary

Fibroblasts

OCCs

ES cells

150

100

50

0

minus50

minus100 minus50 0 50 100

LONBCjRONBCj

ESCcjes001P96

ESCcjes001P91

ESCcjes001P94ESCcjes001P83

P4T1-C1

P4T2-C1

CjFibs4CjFibscj-11CjFibscj-19

CjFibscj-12CjFibscj-20CjFibscj-3

(a)

LOC100411838

LOC100399713RNF17LOC100394179LOC100414988LOC100414988ENSCJAT000000377PRAMETEX12LOC100388137LOC100388137ENSCJAT000000068LOC100412246LOC100408598CP19A_CALJALOC100399315LOC100415170LOC100385174ENSCJAT000000377LOC100402230TDRD1CPVLLOC100398487ENSCJAT000000316

ENSCJAT000000333

ENSCJAT000000532

TDRD1LOC100392679CPVLCPVLGPAT2

SPINK2AMHR2SPINK2LOC100397701TDRD9STK31HBB_CALJAHBB_CALJALOC100400731LOC100400032B0VX78_CALJAHBB_CALJALOC100400731

LOC100391376

LOC100411838P79159_CALJAMAELSYCP2LMAEL

Ovary versus OCCs top 50

Ovary OCCsColor key

0 2Row Z-score

(b)

LOC100398751

LOC100405099LOC100387244

LOC100402277LOC100402277LOC100402277LOC100412434LOC100387684LOC100405924LOC100409409

LOC100407886

LOC100402066

LOC100412217LOC100412217LOC100412217

KRT36GC

VCAM1VCAM1

LOC100393188

ENSCJAT000000598ENSCJAT000000139ENSCJAT000000047LYSC_CALJALECT1

ENSCJAT000000433ENSCJAT000000333ENSCJAT000000008ENSCJAT000000303

ENSCJAT000000440CTSE

ENSCJAT000000574ENSCJAT000000385

ENSCJAT000000054

ENSCJAT000000403ENSCJAT000000581ENSCJAT000000612ENSCJAT000000104ENSCJAT000000070ENSCJAT000000252ENSCJAT000000020ENSCJAT000000277ENSCJAT000000410


COL2A1COL2A1COL2A1COL2A1COL2A1

OCCs versus fibroblasts top 50

OCCs fibroblastsColor key

0 2Row Z-score(c)

LOC100400589LOC100405014LOC100384946LOC100398697

LOC100408978

LOC100396888DDX43LOC100395243LOC100398697PECAM1LOC100384946LOC100413304LOC100408978LOC100408978LOC100408978LOC100408978LOC100406610LOC100410228SLC13A5

CDH1CDH1GYLTL1BCDH1

TDRD1

GDF3PDPN

LOC100386871

LOC100390443LOC100396000


LOC100384946LOC100399586NLRP7NLRP7CD48ENSCJAT000000333ENSCJAT000000333

ENSCJAT000000434

ENSCJAT000000434ENSCJAT000000218ENSCJAT000000525

ENSCJAT000000159


OCCs versus ES cells top 50

OCCs ES cellsColor key

0 2Row Z-score(d)

Figure 2 Continued


Cell adhesionIon transport

Integrin-mediated signaling pathwayMulticellular organismal development

G-protein coupled receptor signaling pathwayCell-matrix adhesion

Calcium ion transportHomophilic cell adhesion

Neurotransmitter transportRegulation of peptidyl-tyrosine phosphorylation

0 5 10 15 20 25

minuslog10(FDR)

(e)

Figure 2 Transcriptome analysis of OCCs (a) Principal component analysis of the transcriptome analyses of ovarian cell colonies (OCCs)neonatal ovaries which served as starting material for OCC cultures embryonic stem cells and fibroblastsThe latter two served as referencesamples OCC transcriptomes differ from the neonatal ovariesrsquo transcriptomes However ovaries ES cells and OCCs are more similar amongeach other than to fibroblasts (see component 1 relative weight of 3854) (b) Top 50 differentially expressed genes between native neonatalovary and OCCs For gene bank and Ensembl identifiers see Table 1 of Supplementary Material (c) Top 50 differentially expressed genesbetween OCCs and fibroblasts (d) Top 50 differentially expressed genes between OCCs and ES cells (e) Gene ontology analysis of OCCsversus native ovary Cell adhesion ion and neurotransmitter transporters and signaling pathways are predominantly upregulated in OCCscompared to the native ovary

other cadherins such as CDH2 (at similar levels in culturedovarian cells compared to ovaries fibroblasts and ES cells)and CDH22 (the same range as neonatal ovaries 10ndash20of fibroblasts and 50 of ES cells) were expressed by theOCCs Hence the phenotypical and molecular indicatorsof the status of the cells constituting the OCCs are notcongruent In order to initially characterize the features ofthe OCCs we performed a gene ontology analysis based onthe genes upregulated in OCCs compared to native ovary(Figure 2(e)) This shows that particularly cell adhesion ionand neurotransmitter transport and signaling pathways areupregulated in the OCCs

In summary the transcriptome data indicate that germcells are absent from theOCCsHowever the detailed identityof the OCCs remains unclear so far Due to the limitednumber of samples (119899 = 2) and the fact that we couldanalyze only one time point by deep sequencing we furtherinvestigated specific genes by RT-qPCR in different passagesto obtain also longitudinal data over the course of the OCCculture

We have recently shown that the neonatal marmosetmonkey ovary contains primitive proliferating germ cellsexpressing the germ cell and pluripotency markers OCT4ASALL4 LIN28 and the general germ cell marker VASA(DDX4) [18] All these markers are only very poorly repre-sented in the transcriptomes of the early passage OCCs orwere even absent We also failed to detect OCT4A LIN28or VASA on the protein level in early passage OCC samplesby a well-established immunohistochemistry protocol [18](data not shown) In order to quantify the expression ofselected key marker genes in OCCs in relation to ES cellsskin fibroblasts and neonatal ovaries by an independentmethod and also at higher (gt4) passages we performed RT-qPCR for a number of pluripotency and (premeiotic) germ

cell markers including OCT4A [25] NANOG [25] SALL4[26] LIN28 [27] VASA [28 see also Figure 5] DAZL [29]NOBOX [30] DPPA3STELLAPGC7 [31 32] PRDM1 [33]and PRDM14 [34 35] Figure 3 shows the exemplary RT-qPCR data of one culture from passage 1 to passage 13These data confirm that the most indicative pluripotencyfactors OCT4A and NANOG were not expressed in all OCCsamples analyzed (Figure 3) In contrast SALL4 LIN28 andVASA were induced in passages geP4 except for SALL4 andLIN28 in P9 In order to further substantiate these findingswe also tested the expression of PRDM1 PRDM14 DAZLDPPA3 NOBOX and SCP3 in the OCCs from differentpassages (Figure 4) DAZL DPPA3 NOBOX and SCP3 asspecific germ cell genes were absent or very low at lowpassages which is in concordance with the data shown inFigure 3 In later stages however all markers were detectableat variable levels In contrast PRDM1 and PRDM14 earlygerm cell specification genes but not specific to germ cellswere robustly expressed in all cell culture samples Thesedata suggest that our culture supports the survival andprobably also the selection of cells that have the potential to(re)express a set of premeiotic and female germ cell markergenes

33 The OCCs Generate Oocyte-Like Cells OCCs generatedlarge free-floating spherical cells which we termed oocyte-like cells (OLCs [17]) OLCs had a morphology resemblingimmature oocytesTheir diameter ranged from 20 to sim40 120583m(Figure 5(a)) We sometimes also observed small structuresslightly resembling polar bodies (Figure 5(a) right picture)However we never observed Zona pellucida nor follicle-like structures as described by Dyce et al [17] For com-parison of the OLCs with real marmoset monkey oocytessee Figure 5(b) Importantly OLCs developed even after 20


002040608

11214

OCT4

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(a)

002040608

11214

NANOG

2minusΔΔ

CT

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

(b)

002040608

112

SALL4

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(c)

002040608

112

LIN28

2minusΔΔ

CT

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

(d)

0

05

1

15VASA

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(e)

Figure 3 RT-qPCR analysis of OCCs in different passages Real-time quantitative RT-PCR analysis of selected key pluripotency and germcell markers in OCCs OCT4 NANOG SALL4 and LIN28 are all robustly expressed in pluripotent stem cells as well as in primitive germcells In contrast OCT4 and NANOG are absent from OCCs SALL4 and LIN28 were absent or very low at low passages and increased inexpression at higher passages VASA is a general germ cell marker and was detected in later OCC passages and in the neonatal ovary

passages and more than 5 months of culture but were notobserved in passages lt 4 We also observed OLCs neitherin MEF-only cell cultures nor in ESC cultures which arealso based on MEFs This strongly indicates that the OLCsindeed derive from the OCCs To initially characterize theOLCs we tested the expression of key pluripotency andgerm cell markers by RT-qPCR We collected 28 OLCs andrandomly allocated the cells to one of two groups (termedOLCs1 and OLCs2) which were then analyzed OCT4ANANOG and LIN28 were low in OLCs compared to EScells and neonatal ovaries (Figures 5(c)ndash5(e)) SALL4 wasrobustly expressed (Figure 5(f)) in the range of the controlsIn contrast PRDM14 DPPA3 DAZL and VASA were muchhigher in OLCs than in neonatal ovary indicating very robustexpression of these germ cell markers in OLCs (Figures 5(g)ndash5(j))NOBOX was in a similar range as in the neonatal ovaryImportantlyDAZLVASA andNOBOX are specific germ line

markers and are all not expressed by ES cells and fibroblasts(Figures 5(i)ndash5(k)) As a marker of meiosis we also testedSCP3 This mRNA was also detected in OLCs although atlower levels compared to the neonatal ovary (Figure 5(l))SCP3 was undetectable in ES cells and fibroblasts A goodindicator of meiosis is chromatin condensation in prepara-tion of the reduction division (Figure 5(m) left) Howeverwe did not see a comparable chromatin condensation inOLCs In contrast the OLCs showed a homogenous DAPIsignal throughout the nucleus (Figure 5(m) right) Althoughthere was no evidence for meiosis the expression of themarkers strongly indicates a germ cell identity of the OLCsIn order to further corroborate the germ line identity ofthe OLCs we aimed at detecting also VASA protein inthe OLCs First we characterized the VASA antibody toprevent misleading antibody-based results as we [36] andothers [37] recently described In western blot analysis a


002040608

11214

PRDM1

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(a)

002040608

11214

PRDM14

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(b)

002040608

112

DAZL

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(c)

002040608

112

DPPA3

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(d)

NOBOX

002040608

112

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(e)

SCP3

002040608

11214

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(f)

Figure 4 Expression of additional germ cell markers in OCCs PRDM1 and PRDM14 are necessary for germ cell specification and areexpressed in all OCC passages in the range of the controlsDAZL andDPPA3 are also necessary for germ cell specification andwere detectableonly in few samplesNOBOX is a female-specific germ cell marker andwas detectable at relatively low levels inmost samples SCP3 is ameiosismarker and was detectable only in some samples at variable levels In general the germ-cell-specific markers were very low or absent duringthe first three passages

very intense and prominent band of the expected size ofsim85 kDa was detected in testis indicating a high specificityof the VASA antibody (Figure 6(a)) Lack of a VASA signalin the ovary was due to the fact that the ovary was froman old monkey with an almost exhausted ovarian germ cellreserve Hence VASA was below the detection limit in theprotein homogenate of the aged ovary We also tested theVASA antibody in immunohistochemistry on tissue sectionsfrom adultmarmoset testis and ovary (Figures 6(b) and 6(c))In both sexes the antibody very robustly and specificallydetected an epitope in germ cells resulting in clear cytoplas-mic germ cell labeling The nongerm cells were not stainedor showed only faint background staining These findingsdemonstrate the specificity of the antibody also formarmosetVASA proteinWe then used this antibody to detect potential

OLCs in OCCs When we fixed OCCs of the 5th passagein situ large cells with strongly condensed chromatin asindicated by strong DAPI fluorescence in Figure 6(d) werelabeled by the VASA antibody (Figure 6(e)) Whether thesignal of the surrounding cells is only background staining orwhether it highlights small germ cell progenitor cells cannotbe decided at present The diameter of the large labeledcell was approximately 35 120583m like the diameter of the OLCsshown in Figure 5(a) We also detached the cell cultures fromthe culture dish and processed themmdashlike the testis and ovaryshown in Figures 6(b) and 6(c)mdashfor immunohistochemistryWe detected large isolated cells that were strongly stained forVASA (Figures 6(f) and 6(g)) The control where VASA wasreplaced by the corresponding IgG showed only very faintbackground signals (Figure 6(h)) These data demonstrate


P23P10 P17 P20

(a)

(b)

OCT4

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

002040608

112

2minusΔΔ

CT

(c)

NANOGO

LC1

OLC

2

NB

ESC

Fibr

obla

sts

002040608

11214

2minusΔΔ

CT

(d)

LIN28

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

05

1

15

2minusΔΔ

CT

(e)

SALL4

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

05

1

15

2minusΔΔ

CT

(f)

PRDM14

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

2

4

6

8

10

2minusΔΔ

CT

(g)

DPPA3

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

5

10

15

20

2minusΔΔ

CT

(h)

Figure 5 Continued


DAZL

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

5

10

15

202minusΔΔ

CT

(i)

VASA

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

2

4

6

8

2minusΔΔ

CT

(j)

NOBOX

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts0123456

2minusΔΔ

CT

(k)

SCP3

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

002040608

112

2minusΔΔ

CT

(l)

OocyteDAPI OLCDAPI

(m)Figure 5 Oocyte-like cells derived from OCCs highly express germ cell markers (a) Oocyte-like cells that spontaneously developed even inhigh cell culture passages The cells were freely floating in the cell culture medium and had a diameter of approximately 40 120583mThe passagenumber is indicated in the upper right corner of each picture Sometimes small spherical structures attached to theOLCs could be seen slightlyresembling polar bodies (arrow) (b) Left a group of natural marmosetmonkey oocytes Some oocytes are still associated with granulosa cellsRight highermagnification of amarmosetmonkey oocyte with a robust Zona pellucida (cndashl) Oocyte-like cells express pluripotency and germcell markers Relative mRNA levels of selected pluripotency and germ cell markers in oocyte-like cells as revealed by real-time quantitativeRT-PCR For OCT4A SALL4 and LIN28 ES cells were used as positive control Neonatal ovary was used as positive control for the germcell markers Fibroblasts served as biological negative control (m) DAPI staining of a natural oocyte (left) and of an OLC While the oocyteshows strongly compacted chromatin the DAPI staining of the OLC is homogenous indicating a different chromatin state in OLCs andnatural oocytes

that there are isolatedVASAprotein-positive oocyte-like cellsin the cultures derived from neonatal marmoset monkeyovary

34 The Relative Marker Abundance Is Decreasing within OnePassage To obtain initial information on the transcriptabundance of themarkers during the OCC development overtime within one passage we isolated RNA from duplicatesamples from OCCs after 2 4 6 and 8 days OCT4ANANOG and LIN28 were very low or absent (Figures 7(a)ndash7(c)) In contrast SALL4 and VASA were robustly detectablein all samples (Figures 7(d) and 7(e)) Both markers exhib-ited a high abundance at day 2 Later time points (days

4ndash8) showed a decrease in transcript abundance relativeto GAPDH probably reflecting a higher proliferation rate ofthemarker-negative (butGAPDH expressing) cells comparedto the marker-positive cells leading to a dilution effect of theVASA- and SALL4-positive cells

35 No Production of Sex Steroids by the OCCs We werewondering whether the OCCs have the ability to producefemale sex steroids like specific cells of the ovary in vivoTherefore we tested medium samples from low and highculture passages after several days without medium changeSamples were analyzed for estradiol which is primarilyproduced by ovarian granulosa cells and progesterone which


(kDa)120100

80

60

50

50

40

40

VASA

(DD

X4)

Live

r

Stom

ach

Panc

reas

Skin

Testi

s

120573-a

ctin

Ova

rylowast

(a)

(b) (c)

(d) (e)

50120583m

(f)

50120583m

(g)

50120583m

(h)

Figure 6 Characterization of the VASA antibody and detection of VASA-positive cells in OCCs (a) Western blot analysis using marmosetmonkey protein samples demonstrates the specificity of the VASA antibody lowast indicates that the ovary was from an aged animal The ovariangerm cell pool was therefore most likely strongly reduced or even exhausted leading to undetectable VASA protein concentrations in thesample (b and c) Immunohistochemical application of the VASA antibody to marmoset gonads showed germ-cell-specific labeling in theadult testis and the adult ovary (d) DAPI staining of a part of an OCC Note the intensely stained nucleus in the central part (e) The samearea shown in (d) The large cell containing the intensely stained nucleus strongly stains for VASA The signal is blurry due to the fact thatthe picture was taken through the bottom of a normal cell culture dish (f g) Examples of isolated VASA-positive cells in sections of paraffin-embedded OCCs

is synthetized by the cells of the Corpus luteum Neitherestradiol nor progesterone was detected in medium sam-ples Moreover FSHR transcripts were undetectable in OCCsamples by deep sequencing and LHCGR abundance waslower than in neonatal ovary fibroblasts and ES cells (datanot shown) Hence the colonies do not consist of functionalendocrine cells of the ovary

36 Neither TeratomaNor Ovarian Tissue Formation in a Sub-cutaneous Transplantation Assay Subcutaneous transplanta-tion of cells into immune-deficient mice is a useful approachto assay cells with regard to their differentiation capabilitiesfor example the teratoma formation assay Moreover wehave recently shown that single-cell suspensions derived from

dissociated neonatal monkey testis can reconstitute complextestis tissue after transplantation into immune-deficient mice[24] In order to test whether the OCCs have the abilityto form ovarian tissue under the ldquoin vivordquo conditions aftertransplantation we injected sim106 cells per mouse from the5th passage subcutaneously into 4 female adult NOD-SCIDmice Tissues from the injection site were collected for his-tological analysis after 15 weeks Neither ovary-like tissue norteratoma or any other conspicuous tissue was found (data notshown)

4 Discussion

A controversial debate on the presence of mitotically activegerm line stem cells in the postnatal ovary characterized


D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

NANOG

Fibr

obla

sts

002040608

11214

2minusΔΔ

CT

(a)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

OCT4

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(b)LIN28

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(c)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

SALL4

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(d)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

VASAFi

brob

lasts

0

02

04

06

08

1

12

2minusΔΔ

CT

(e)

Figure 7 Pluripotency and germ cell markermRNA abundance during the development of OCCswithin one passage as revealed by real-timequantitative PCR Positive controls for OCT4A SALL4 and LIN28 were ES cells Neonatal ovary was used as positive control for the germcell marker VASA NANOG was very low and OCT4A and LIN28 were absent at all time points analyzed Relative abundance of SALL4 andVASA generally decreased with time during the culture period D days of culture W well for cell culture NB newborn ovary

the last decade of ovarian germ cell research in mammalsSeveral reports provided data supporting the presence ofovarian germ line stem cells in postnatal mouse ovaries forexample [10 13 38] while other studies failed to identifyfemale germ line stem cells for example [7 8] therebysupporting the classical view of ovarian biology [1] Inprimates including humans the published data are similarlycontroversial and still not fully conclusive Byskov et al [3]failed to detect oogonia which may be candidate cells for

ovarian stem cells in the postnatal human ovary older thantwo years and even in younger postnatal ovaries the stem cellmarker OCT4 was detectable only very rarely We recentlyalso failed to detect pluripotency and stem cell marker-positive cells in marmoset ovaries at one year of age [18] Onthe other hand White et al [12] isolated mitotically activecells from human adult ovarian cortex that had the potentialto form oocyte-like cells in vitro and to form ovarian folliclesin a combined alloxenografting approach Recently however


Yuan et al [9] failed to provide evidence for mitotically activegerm line stem cells in rhesus monkeys (Macaca mulatta)and mice concluding that adult ovaries do not undergo germcell renewal Furthermore almost ten years ago Dyce et al[17] reported that not only ovarian cells have the potentialto develop female germ cells but also fetal porcine skincells have They formed oocyte-like cells in vitro which wereextruded from hormone-responsive follicle-like aggregates

We have established a long-term cell culture system forneonatalmarmosetmonkey ovarian cells Seeding of themar-moset monkey ovarian cell suspension onto the MEF cellsresulted in the development of cell colonies morphologicallyresembling colonies which have been observed previously bydifferent other groups for human [39] and mouse [38 40]ovarian cell cultures However while these studies reportedthe robust expression of pluripotency markers such as OCT4and NANOG and therefore claimed an ES cell-like characterof the cells [40] we failed to detect these core pluripotencymarkers in marmoset ovarian cell cultures on the transcriptand on the protein level (the latter not shown) althoughour starting material that is neonatal ovary was positivefor these factors In fact marmoset monkey primordial germcells [19] and oogonia [18] robustly express OCT4 Thisdiscrepancy between the native premeiotic germ cells and thecultured ovarian cells shows that themarmoset OCCs did notcontain remaining (ldquocontaminatingrdquo) primordial germ cellsor oogonia All our data point to the fact that premeioticgerm cells were absent from the early passages of the culturedovarian cells This is also in contrast to a publication on pigovarian stem cells Bui and colleagues [41] derived putativestem cells from adult pig ovary that gave rise to germ cell-like cells corresponding to primordial germ cells Howeverthese cells unlike the cells described in our present studyalso robustly expressed pluripotency factors such as OCT4and NANOG Moreover the phenotype of the pig cells inculture was different from the colonies observed in this studyand other studies for example [40] Interestingly howeverdespite the clearly different starting conditions both our andBuirsquos cell culture systems resulted in the development of largeoocyte-like cells

In addition to the absence of the pluripotency factors wedid not detect even a single DAZL MAEL RNF17 TEX12TEX101 or TDRD1 transcript in OCCs of the 4th passage bydeep sequencing while all these germ cell transcripts wereabundant in the neonatal ovary samples further supportingthe absence of germ cells from the marmoset ovarian cellcultures at low passages Hence we conclude that marmosetmonkey OCCs are neither germ cells nor pluripotent stemcellsThe exact identity of theOCCswill be analyzed in futurestudies However despite showing morphology resemblingan epithelium the OCCs lacked characteristic proteins ofepithelia like E-cadherin and cytokeratins However fromOCC passage 4 onwards we observed the development ofindividual oocyte-like cells strongly expressing the germcell genes VASA DAZL NOBOX DPPA3 and SALL4 Fur-thermore also the meiotic marker SCP3 was expressed inOLCs even though at moderate levels In contrast OCT4ANANOG and LIN28 were low in OLCs In addition to themarker expression data on the mRNA level we wanted to

confirm VASA also on the protein level We detected robustVASA protein expression in OLCs Altogether this markerprofile suggests that the OLCs may correspond to a latepremeiotic stage These characteristics are partly similar tothose of the OLCs described by Dyce et al [17] HoweverDyce and colleagues showed that OLCs developed from cellaggregates that detached from the cell culture surface andformed hormone-responsive follicle-like structures Thenthe OLCs were extruded from the follicles and released intothe medium From sim500000 skin cells approximately 6ndash70large cells were extruded Our findings were in the samerangewith typically 5ndash10 largeOLCs perwell and passage Butwe never observed follicle-like structures Moreover we didnot obtain any evidence for an endocrine regulation of OLCdevelopment in the marmoset monkey ovarian cell cultures

Since we originally intended to culture oogonia we spundown the cells down primary cells at 200 g This may beinsufficient to quantitatively collect those cells in the pelletthat were cultured in previous studies where the ovariangerm line stem cells were sedimented at 300 g [13] or 1000 g[14] respectively Based on this we hypothesize that wecollected in the present study oogonia (and other largersomatic cells) using a force of 200 g but only marginalamounts of the progenitors of the OLCs which probably area subpopulation of the OSE [14] The oogonia apparentlydid not survive in our culture system Therefore we wereunable to detect pluripotency markers in our culture andgerm cell markers in the early passages However we furtherhypothesize that despite the low 119892 force applied in our studysomeOSE cells with stem cell capacity were still present in theculture They were hypothetically able to develop into OLCsafter more than three passages

We also tested magnetic activated cell sorting (MACS)to enrich putative stem cells However MACS using forexample TRA-1-81 antibodies was not successful It mustbe noted however that the neonatal marmoset ovary isextremely tiny and that its availability is very limited Indeedthe size of the neonatal ovary is only about 2mm times 1mm times1mm Hence experimental refinement and cell enrichmentapproaches are extraordinarily challenging Therefore wedecided in the present study to culture the unsorted cellpopulation obtained after tissue digestion

In summary we have established a long-term neonatalmarmoset monkey ovarian cell culture system However wefailed to detect pluripotent stem and premeiotic germ cellmarkers in low OCC passages From passage 4 onwardshowever OLCs developed and were still developing at highpassages (gt20) after more than 5 months of culture Con-sidering the marker expression data the view of a germcell identity of the OLCs appears justified An essentialprerequisite of gamete formation is meiosis [42] Except forthe expression of SCP3 which is an essential protein formeiotic synaptonemal complex formation we obtained noevidence for meiotic entry of the OLCs except the formationof some structures morphologically resembling polar bodiesWe also never observed a Zona pellucida Altogether OLCsgenerated in this culture system appear to be germ line cellsbut they are neither functional female gametes nor do theyrepresent meiotic germ cell stages


Currently it remains to be proven from which progenitorcells the OLCs develop in our culture system So far we werenot able to identify these cells in the marmoset However acandidate tissue for the presence of ovarian stem cells withgerm line potential could be the ovarian surface epitheliumwhich is discussed to be a multipotent tissue possibly harbor-ing a stem or progenitor cell type [43] In this regard also theconcept of the very small embryonic-like stem cells (VSELs)should be taken into account [16]

5 Conclusion and Outlook

In conclusion we have established a nonhuman primatecell culture system that allows the long-term culture anddevelopment of OLCs from a nonhuman primate speciesThe vast majority of the cultured cells in this study how-ever are neither pluripotent stem cells nor germ (linestem) cells as has been suggested or shown in previousreports [38ndash40] Future experiments should aim at resolvingthis discrepancy and test whether this is a species-specificphenomenon

Future experiments will also aim at the identificationand functional characterization of the stemprogenitor cellpopulation in the marmoset culture system Furthermoreprotocols are needed that support the entry of the OLCsinto meiosis potentially giving rise to mature oocytes in thefuture Besides functional testing of these cells their correctepigenetic state needs to be confirmed In the future a refinedculture system based on the one described here may allowmore detailed in vitro studies on early phases of primate germcell development and on the molecular and cellular identityofOLCs and their progenitors in an experimentally accessibleNHP system In the long term this may also contribute in thefuture to the development of novel therapeutic approaches offemale infertility

Conflict of Interests

The authors declare that there is no conflict of interests thatcould be perceived as prejudicing the impartiality of theresearch reported

Authorsrsquo Contribution

Bentolhoda Fereydouni carried out conception and designcollection and assembly of data data analysis and interpre-tation paper writing and final approval of paper GabrielaSalinas-Riester carried out collection and assembly of datadata analysis and interpretation paper writing and finalapproval of paper Michael Heistermann carried out col-lection and assembly of data data analysis and interpreta-tion and final approval of paper Ralf Dressel carried outfinancial support collection andor assembly of data andfinal approval of paper Lucia Lewerich carried out collectionandor assembly of data data analysis and interpretationand final approval of paper Charis Drummer carried outprovision of study material and final approval of paperRudiger Behr carried out conception and design financialsupport collection andor assembly of data data analysis

and interpretation paper writing and final approval ofpaper

Acknowledgments

The authors appreciate the excellent assistance and thesupport of Nicole Umland Angelina Berenson and GescheSpehlbrink They are also very thankful to Erika Sasakifor providing the ES cells Katharina Debowski for ES cellculture and the members of the animal facility for excellentanimal husbandryThey thank EllenWiese for administrativesupport The support of Bentolhoda Fereydouni by thescholarship of the Deutscher Akademischer Austauschdienst(DAAD) is gratefully acknowledged Bentolhoda Fereydouniis on a scholarship of the Deutscher Akademischer Aus-tauschdienst (DAAD) The work was mainly supported byinstitutional resources and marginally by a grant of theDeutsche Forschungsgemeinschaft (DFG Be2296-6) entitledldquoPluripotent cells from marmoset testisrdquo to Rudiger BehrThe cell injection assays in mice performed by Ralf Dresselwere supported by a Collaborative Research Centre from theDeutsche Forschungsgemeinschaft (SFB1002 TP C05)

References

[1] S Zuckerman ldquoThe number of oocytes in the mature ovaryrdquoRecent Progress in Hormone Research vol 6 pp 63ndash108 1951

[2] A G Byskov M J Faddy J G Lemmen and C Y AndersenldquoEggs foreverrdquo Differentiation vol 73 no 9-10 pp 438ndash4462005

[3] A G Byskov P E Hoslashyer C Yding Andersen S G KristensenA Jespersen and KMoslashllgard ldquoNo evidence for the presence ofoogonia in the human ovary after their final clearance duringthe first two years of liferdquo Human Reproduction vol 26 no 8pp 2129ndash2139 2011

[4] Y Liu C Wu Q Lyu et al ldquoGermline stem cells and neo-oogenesis in the adult human ovaryrdquo Developmental Biologyvol 306 no 1 pp 112ndash120 2007

[5] E Bendsen A G Byskov C Y Andersen and L G Wester-gaard ldquoNumber of germ cells and somatic cells in human fetalovaries during the first weeks after sex differentiationrdquo HumanReproduction vol 21 no 1 pp 30ndash35 2006

[6] H Stoop F Honecker M Cools R de Krijger C Bokemeyerand L H J Looijenga ldquoDifferentiation and development ofhuman female germ cells during prenatal gonadogenesis animmunohistochemical studyrdquoHumanReproduction vol 20 no6 pp 1466ndash1476 2005

[7] H Zhang W Zheng Y Shen D Adhikari H Ueno and KLiu ldquoExperimental evidence showing that no mitotically activefemale germline progenitors exist in postnatal mouse ovariesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 109 no 31 pp 12580ndash12585 2012

[8] L Lei and A C Spradling ldquoFemale mice lack adult germ-line stem cells but sustain oogenesis using stable primordialfolliclesrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 110 no 21 pp 8585ndash8590 2013

[9] J Yuan D Zhang L Wang et al ldquoNo evidence for neo-oogenesis may link to ovarian senescence in adult monkeyrdquoStem Cells vol 31 no 11 pp 2538ndash2550 2013


[10] J Johnson J Canning T Kaneko J K Pru and J L TillyldquoGermline stem cells and follicular renewal in the postnatalmammalian ovaryrdquo Nature vol 428 no 6979 pp 145ndash1502004

[11] J Johnson J Bagley M Skaznik-Wikiel et al ldquoOocyte genera-tion in adult mammalian ovaries by putative germ cells in bonemarrow and peripheral bloodrdquo Cell vol 122 no 2 pp 303ndash3152005

[12] Y A R White D C Woods Y Takai O Ishihara H Sekiand J L Tilly ldquoOocyte formation by mitotically active germcells purified from ovaries of reproductive-age womenrdquo NatureMedicine vol 18 no 3 pp 413ndash421 2012

[13] K Zou Z Yuan Z Yang et al ldquoProduction of offspring from agermline stem cell line derived from neonatal ovariesrdquo NatureCell Biology vol 11 no 5 pp 631ndash636 2009

[14] A Bukovsky M Svetlikova and M R Caudle ldquoOogenesis incultures derived from adult human ovariesrdquo Reproductive Biol-ogy and Endocrinology vol 3 article 17 2005

[15] I Virant-Klun N Zech P Rozman et al ldquoPutative stem cellswith an embryonic character isolated from the ovarian surfaceepithelium of women with no naturally present follicles andoocytesrdquo Differentiation vol 76 no 8 pp 843ndash856 2008

[16] S Parte D Bhartiya J Telang et al ldquoDetection characteri-zation and spontaneous differentiation in vitro of very smallembryonic-like putative stem cells in adult mammalian ovaryrdquoStem Cells and Development vol 20 no 8 pp 1451ndash1464 2011

[17] P-WDyce LWen and J Li ldquoIn vitro germline potential of stemcells derived from fetal porcine skinrdquoNature Cell Biology vol 8no 4 pp 384ndash390 2006

[18] B Fereydouni C Drummer N Aeckerle S Schlatt and RBehr ldquoThe neonatal marmoset monkey ovary is very primitiveexhibitingmany oogoniardquoReproduction vol 148 no 2 pp 237ndash247 2014

[19] N Aeckerle C Drummer K Debowski C Viebahn and RBehr ldquoPrimordial germ cell development in the marmosetmonkey as revealed by pluripotency factor expression sugges-tion of a novel model of embryonic germ cell translocationrdquoMolecular Human Reproduction vol 21 no 1 pp 66ndash80 2015

[20] T Muller G Fleischmann K Eildermann et al ldquoA novelembryonic stem cell line derived from the common marmosetmonkey (Callithrix jacchus) exhibiting germ cell-like character-isticsrdquoHuman Reproduction vol 24 no 6 pp 1359ndash1372 2009

[21] K Debowski R Warthemann J Lentes et al ldquoNon-viralgeneration of marmoset monkey iPS cells by a six-factor-in-one-vector approachrdquo PLoS ONE vol 10 no 3 2015

[22] M Heistermann S Tari and J K Hodges ldquoMeasurement offaecal steroids for monitoring ovarian function in new worldprimates callitrichidaerdquo Journal of Reproduction and Fertilityvol 99 no 1 pp 243ndash251 1993

[23] M Heistermann M Finke and J K Hodges ldquoAssessment offemale reproductive status in captive-housed Hanuman langurs(Presbytis entellus) by measurement of urinary and fecal steroidexcretion patternsrdquoAmerican Journal of Primatology vol 37 no4 pp 275ndash284 1995

[24] N Aeckerle R Dressel and R Behr ldquoGrafting of neonatal mar-moset monkey testicular single-cell suspensions into immun-odeficient mice leads to ex situ testicular cord neomorphogen-esisrdquo Cells Tissues Organs vol 198 no 3 pp 209ndash220 2013

[25] R M Perrett L Turnpenny J J Eckert et al ldquoThe earlyhuman germ cell lineage does not express SOX2 during in vivodevelopment or upon in vitro culturerdquo Biology of Reproductionvol 78 no 5 pp 852ndash858 2008

[26] K EildermannNAeckerle KDebowski et al ldquoDevelopmentalexpression of the pluripotency factor sal-like protein 4 in themonkey human and mouse testis restriction to premeioticgerm cellsrdquo Cells Tissues Organs vol 196 no 3 pp 206ndash2202012

[27] N Aeckerle K Eildermann C Drummer et al ldquoThe pluripo-tency factor LIN28 in monkey and human testes a marker forspermatogonial stem cellsrdquo Molecular Human Reproductionvol 18 no 10 Article ID gas025 pp 477ndash488 2012

[28] D H Castrillon B J Quade T Y Wang C Quigley and CP Crum ldquoThe human VASA gene is specifically expressed inthe germ cell lineagerdquo Proceedings of the National Academy ofSciences of the United States of America vol 97 no 17 pp 9585ndash9590 2000

[29] Y Lin M E Gill J Koubova and D C Page ldquoGerm cell-intrinsic and -extrinsic factors govern meiotic initiation inmouse embryosrdquo Science vol 322 no 5908 pp 1685ndash16872008

[30] A Rajkovic S A Pangas D Ballow N Suzumori and M MMatzuk ldquoNOBOXdeficiency disrupts early folliculogenesis andoocyte-specific gene expressionrdquo Science vol 305 no 5687 pp1157ndash1159 2004

[31] Y-J Liu T Nakamura and T Nakano ldquoEssential role of DPPA3for chromatin condensation inmouse oocytogenesisrdquoBiology ofReproduction vol 86 no 2 article 40 2012

[32] M Sato T Kimura K Kurokawa et al ldquoIdentification of PGC7a new gene expressed specifically in preimplantation embryosand germ cellsrdquoMechanisms of Development vol 113 no 1 pp91ndash94 2002

[33] Y Ohinata B Payer D OrsquoCarroll et al ldquoBlimp1 is a criticaldeterminant of the germ cell lineage in micerdquo Nature vol 436no 7048 pp 207ndash213 2005

[34] M Yamaji Y Seki K Kurimoto et al ldquoCritical function ofPrdm14 for the establishment of the germ cell lineage in micerdquoNature Genetics vol 40 no 8 pp 1016ndash1022 2008

[35] F Sugawa M J Arauzo-Bravo J Yoon et al ldquoHuman primor-dial germ cell commitment in vitro associates with a uniquePRDM14 expression profilerdquoThe EMBO Journal vol 34 no 8pp 1009ndash1024 2015

[36] R Warthemann K Eildermann K Debowski and R BehrldquoFalse-positive antibody signals for the pluripotency factorOCT4A (POU5F1) in testis-derived cells may lead to erroneousdata and misinterpretationsrdquo Molecular Human Reproductionvol 18 no 12 pp 605ndash612 2012

[37] R Ivell K Teerds and G E Hoffman ldquoProper applicationof antibodies for immunohistochemical detection antibodycrimes and how to prevent themrdquo Endocrinology vol 155 no3 pp 676ndash687 2014

[38] J Pacchiarotti C Maki T Ramos et al ldquoDifferentiation poten-tial of germ line stem cells derived from the postnatal mouseovaryrdquo Differentiation vol 79 no 3 pp 159ndash170 2010

[39] M Stimpfel T Skutella B Cvjeticanin et al ldquoIsolation char-acterization and differentiation of cells expressing pluripo-tentmultipotent markers from adult human ovariesrdquo Cell andTissue Research vol 354 no 2 pp 593ndash607 2013

[40] S P Gong S T Lee E J Lee et al ldquoEmbryonic stem cell-like cells established by culture of adult ovarian cells in micerdquoFertility and Sterility vol 93 no 8 pp 2594e9ndash2601e9 2010

[41] H-T Bui N VanThuan D-N Kwon et al ldquoIdentification andcharacterization of putative stem cells in the adult pig ovaryrdquoDevelopment vol 141 no 11 pp 2235ndash2244 2014


[42] M A Handel J J Eppig and J C Schimenti ldquoApplying lsquogoldstandardsrsquo to in-vitro-derived germ cellsrdquo Cell vol 157 no 6pp 1257ndash1261 2014

[43] A Bukovsky M R Caudle I Virant-Klun et al ldquoImmunephysiology and oogenesis in fetal and adult humans ovarianinfertility and totipotency of adult ovarian stem cellsrdquo BirthDefects Research Part C Embryo Today Reviews vol 87 no 1pp 64ndash89 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


the ability to reconstitute oogenesis and produce offspringafter transplantation Other authors reported the productionof oocyte-like cells in vitro from cultures of adult ovarian sur-face epithelium (OSE) [14ndash16] In summary currently thereare several apparently contradictory reports which providedata supporting or negating generation of new oocytes in thepostnatal rodent and primatemdashincluding the humanmdashovaryAdding to this complex situation Dyce et al [17] reportedthat even fetal porcine skin cells can form oocyte-like cells(OLCs) in vitro

We use the common marmoset monkey (Callithrix jac-chus) as a nonhuman primate model to study reproductiveand stem cell biology In contrast to other species usedin reproductive biology the marmoset monkey still hasnumerous oogonia in the neonatal ovary which robustlyexpress the pluripotency associated markers OCT4A LIN28and SALL4 the germ cell marker VASA and the proliferationmarker KI-67 [18] Therefore these oogonia in the neonatalmarmoset ovary share important pluripotency markers withmarmoset monkey embryonic stem (ES) cells [19] We failedto detect this proliferating and pluripotency-marker-positivecell population in the one-year-old and adult ovary [18]indicating a fast postnatal oogonial clearing also in thisnonhuman primate species Here we report studies onthe culture of neonatal marmoset ovarian cells We origi-nally aimed at culturing the proliferating marmoset monkeyoogonia exhibiting a pluripotency signature similar to mar-moset monkey embryonic stem cells Hence we establisheda mouse embryonic fibroblast- (MEF-) based cell culturesystem which allowed the long-term culture of ovarian cellsHowever germ cell and pluripotency marker expression waslost in the first passages indicating the complete loss of theoogonia After a few passages however germ cell markerexpression recovered and we observed in later passages thedevelopment of large spherical free-floating cells which wecalled oocyte-like cells (OLCs) due to their morphologicalresemblance with the cells reported by Dyce et al [17] OLCshad a diameter up to sim40 120583m and strongly expressed severalgerm cell markers This study demonstrates the developmentof oocyte-like cells in long-term ovarian cell cultures froma translational and experimentally accessible nonhumanprimate species

2 Materials and Methods

21 Animals Marmoset monkeys (Callithrix jacchus) for thisstudy were obtained from the self-sustaining breeding colonyof the German Primate Center (Deutsches Primatenzen-trum DPZ) The German Primate Center is registered andauthorised by the local and regional veterinary governmentalauthorities (Reference number 1229103311900 PK LandkreisGottingen) Health and well-being of the animals werecontrolled daily by experienced veterinarians and animalcare attendants The legal guidelines for the use of animalsand the institutional guidelines of the DPZ for the careand use of marmoset monkeys were strictly followed Theanimals were pair-housed in a temperature- (25 plusmn 1∘C)and humidity-controlled (65 plusmn 5) facility Illumination wasprovided by daylight and additional artificial lighting on

a 1200 1200-hour light dark cycleThe animals were fed adlibitum with a pelleted marmoset diet (ssniff SpezialdiatenSoest Germany) In addition 20 g mash per animal wasserved in the morning and 30 g cut fruits or vegetablesmixed with noodles or rice was supplied in the afternoonFurthermore once per week mealworms or locusts wereserved in order to provide adequate nutrition Drinkingwaterwas always available

In captivitymarmosets sometimes give birth to triplets oreven quadruplets However the mother is usually able to feedand rear only two neonates which is the normal litter size offree-living marmosets Therefore the neonates from tripletbirths were used to collect organs for this study Marmosetmonkey ovaries were obtained from six neonatal animals(postnatal days 1ndash5) All animals were narcotized with Pento-barbital (Narcoren 005mL intramuscular) and euthanizedby an experienced veterinarian with an intracardial injectionof 05mL Pentobarbital before a lack of nourishment causedsuffering of the animals Wherever applicable the ARRIVEguidelines were followed

22 Numbers of Animals Altogether ovaries from 6 neonatalmarmosets were used in this study 5 pairs for culture and onepair as reference for transcriptome analysis

23 Neonatal Common Marmoset Monkey Ovarian Cell Cul-tures Neonatal ovaries (approximate dimensions 2mm times1mm times 1mm see also [18]) were first collected and washedin a Petri dish containing cold DPBS Fat and blood cellswere removed and the whole ovary including the OSE wastransferred to fresh buffer and minced with sterile scissorsMinced ovaries were then transferred to a 15mL falcon tubeAfter gentle centrifugation DPBS was removed and ovariantissue fragments were resuspended in DMEMF12 mediumcontaining collagenase (Sigma C2674) and DNase and keptfor 45 minutes at 37∘C Every 5 to 10min the ovaries weregently pipetted to disintegrate the tissue Finally 10 FBSwas added to inactivate the enzyme In order to removelarger undigested tissue fragments the cells were passedthrough 70 120583m strainer (BD USA) and were centrifuged at200 g for 10min Then the supernatant was removed and thecells were resuspended in culture medium and transferredon a prepared feeder layer of mouse embryonic fibroblast(MEF) feeder at a concentration 3ndash5 times 105 cells per 5 cmwell After one week large colonies were individually pickedwith a microprobe (FST Heidelberg Germany 10032-13)and digested with Accutase (Life Technologies Germany) for4minThen the cells were centrifuged at 300 g for 10 minutesand resuspended in the culture medium for further passagesCultures were maintained at 37∘C under 5 CO

2and 5

oxygen in a humidified incubator

24 Culturing Neonatal Common Marmoset Ovaries Differ-ent cell culture conditions were tested Finally this conditionwas selected DMEMF12 supplemented with FBS (10)PenStrep amphotericin B and human LIF (10 120583gmL) onan irradiated mouse embryonic fibroblast (MEF) feeder celllayer For further passages (usually after 7ndash10 days) colonies
















3 Results




P1 early

(a)

P1 late

(b)

P10

(c)

(d)

200120583m







Component 1 (4270)

Com

pone

nt 2

(23

79

)

Neonatal ovary

Fibroblasts

OCCs

ES cells

150

100

50

0

minus50


LONBCjRONBCj

ESCcjes001P96

ESCcjes001P91


P4T1-C1

P4T2-C1



(a)

LOC100411838


ENSCJAT000000333

ENSCJAT000000532



LOC100391376



Ovary OCCsColor key

0 2Row Z-score

(b)

LOC100398751

LOC100405099LOC100387244


LOC100407886

LOC100402066

LOC100412217LOC100412217LOC100412217

KRT36GC

VCAM1VCAM1

LOC100393188





ENSCJAT000000054






0 2Row Z-score(c)

LOC100400589LOC100405014LOC100384946LOC100398697

LOC100408978


CDH1CDH1GYLTL1BCDH1

TDRD1

GDF3PDPN

LOC100386871

LOC100390443LOC100396000



ENSCJAT000000434


ENSCJAT000000159




0 2Row Z-score(d)

Figure 2 Continued







0 5 10 15 20 25

minuslog10(FDR)

(e)








002040608

11214

OCT4

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(a)

002040608

11214

NANOG

2minusΔΔ

CT

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

(b)

002040608

112

SALL4

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(c)

002040608

112

LIN28

2minusΔΔ

CT

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

(d)

0

05

1

15VASA

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(e)





002040608

11214

PRDM1

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(a)

002040608

11214

PRDM14

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(b)

002040608

112

DAZL

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(c)

002040608

112

DPPA3

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(d)

NOBOX

002040608

112

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(e)

SCP3

002040608

11214

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(f)





P23P10 P17 P20

(a)

(b)

OCT4

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

002040608

112

2minusΔΔ

CT

(c)

NANOGO

LC1

OLC

2

NB

ESC

Fibr

obla

sts

002040608

11214

2minusΔΔ

CT

(d)

LIN28

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

05

1

15

2minusΔΔ

CT

(e)

SALL4

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

05

1

15

2minusΔΔ

CT

(f)

PRDM14

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

2

4

6

8

10

2minusΔΔ

CT

(g)

DPPA3

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

5

10

15

20

2minusΔΔ

CT

(h)

Figure 5 Continued


DAZL

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

5

10

15

202minusΔΔ

CT

(i)

VASA

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

2

4

6

8

2minusΔΔ

CT

(j)

NOBOX

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts0123456

2minusΔΔ

CT

(k)

SCP3

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

002040608

112

2minusΔΔ

CT

(l)

OocyteDAPI OLCDAPI







(kDa)120100

80

60

50

50

40

40

VASA

(DD

X4)

Live

r

Stom

ach

Panc

reas

Skin

Testi

s

120573-a

ctin

Ova

rylowast

(a)

(b) (c)

(d) (e)

50120583m

(f)

50120583m

(g)

50120583m

(h)





4 Discussion



D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

NANOG

Fibr

obla

sts

002040608

11214

2minusΔΔ

CT

(a)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

OCT4

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(b)LIN28

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(c)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

SALL4

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(d)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

VASAFi

brob

lasts

0

02

04

06

08

1

12

2minusΔΔ

CT

(e)






















Acknowledgments


References





















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















3 Results




P1 early

(a)

P1 late

(b)

P10

(c)

(d)

200120583m







Component 1 (4270)

Com

pone

nt 2

(23

79

)

Neonatal ovary

Fibroblasts

OCCs

ES cells

150

100

50

0

minus50


LONBCjRONBCj

ESCcjes001P96

ESCcjes001P91


P4T1-C1

P4T2-C1



(a)

LOC100411838


ENSCJAT000000333

ENSCJAT000000532



LOC100391376



Ovary OCCsColor key

0 2Row Z-score

(b)

LOC100398751

LOC100405099LOC100387244


LOC100407886

LOC100402066

LOC100412217LOC100412217LOC100412217

KRT36GC

VCAM1VCAM1

LOC100393188





ENSCJAT000000054






0 2Row Z-score(c)

LOC100400589LOC100405014LOC100384946LOC100398697

LOC100408978


CDH1CDH1GYLTL1BCDH1

TDRD1

GDF3PDPN

LOC100386871

LOC100390443LOC100396000



ENSCJAT000000434


ENSCJAT000000159




0 2Row Z-score(d)

Figure 2 Continued







0 5 10 15 20 25

minuslog10(FDR)

(e)








002040608

11214

OCT4

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(a)

002040608

11214

NANOG

2minusΔΔ

CT

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

(b)

002040608

112

SALL4

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(c)

002040608

112

LIN28

2minusΔΔ

CT

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

(d)

0

05

1

15VASA

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(e)





002040608

11214

PRDM1

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(a)

002040608

11214

PRDM14

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(b)

002040608

112

DAZL

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(c)

002040608

112

DPPA3

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(d)

NOBOX

002040608

112

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(e)

SCP3

002040608

11214

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(f)





P23P10 P17 P20

(a)

(b)

OCT4

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

002040608

112

2minusΔΔ

CT

(c)

NANOGO

LC1

OLC

2

NB

ESC

Fibr

obla

sts

002040608

11214

2minusΔΔ

CT

(d)

LIN28

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

05

1

15

2minusΔΔ

CT

(e)

SALL4

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

05

1

15

2minusΔΔ

CT

(f)

PRDM14

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

2

4

6

8

10

2minusΔΔ

CT

(g)

DPPA3

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

5

10

15

20

2minusΔΔ

CT

(h)

Figure 5 Continued


DAZL

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

5

10

15

202minusΔΔ

CT

(i)

VASA

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

2

4

6

8

2minusΔΔ

CT

(j)

NOBOX

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts0123456

2minusΔΔ

CT

(k)

SCP3

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

002040608

112

2minusΔΔ

CT

(l)

OocyteDAPI OLCDAPI







(kDa)120100

80

60

50

50

40

40

VASA

(DD

X4)

Live

r

Stom

ach

Panc

reas

Skin

Testi

s

120573-a

ctin

Ova

rylowast

(a)

(b) (c)

(d) (e)

50120583m

(f)

50120583m

(g)

50120583m

(h)





4 Discussion



D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

NANOG

Fibr

obla

sts

002040608

11214

2minusΔΔ

CT

(a)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

OCT4

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(b)LIN28

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(c)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

SALL4

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(d)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

VASAFi

brob

lasts

0

02

04

06

08

1

12

2minusΔΔ

CT

(e)






















Acknowledgments


References





















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


P1 early

(a)

P1 late

(b)

P10

(c)

(d)

200120583m







Component 1 (4270)

Com

pone

nt 2

(23

79

)

Neonatal ovary

Fibroblasts

OCCs

ES cells

150

100

50

0

minus50


LONBCjRONBCj

ESCcjes001P96

ESCcjes001P91


P4T1-C1

P4T2-C1



(a)

LOC100411838


ENSCJAT000000333

ENSCJAT000000532



LOC100391376



Ovary OCCsColor key

0 2Row Z-score

(b)

LOC100398751

LOC100405099LOC100387244


LOC100407886

LOC100402066

LOC100412217LOC100412217LOC100412217

KRT36GC

VCAM1VCAM1

LOC100393188





ENSCJAT000000054






0 2Row Z-score(c)

LOC100400589LOC100405014LOC100384946LOC100398697

LOC100408978


CDH1CDH1GYLTL1BCDH1

TDRD1

GDF3PDPN

LOC100386871

LOC100390443LOC100396000



ENSCJAT000000434


ENSCJAT000000159




0 2Row Z-score(d)

Figure 2 Continued







0 5 10 15 20 25

minuslog10(FDR)

(e)








002040608

11214

OCT4

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(a)

002040608

11214

NANOG

2minusΔΔ

CT

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

(b)

002040608

112

SALL4

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(c)

002040608

112

LIN28

2minusΔΔ

CT

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

(d)

0

05

1

15VASA

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(e)





002040608

11214

PRDM1

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(a)

002040608

11214

PRDM14

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(b)

002040608

112

DAZL

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(c)

002040608

112

DPPA3

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(d)

NOBOX

002040608

112

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(e)

SCP3

002040608

11214

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(f)





P23P10 P17 P20

(a)

(b)

OCT4

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

002040608

112

2minusΔΔ

CT

(c)

NANOGO

LC1

OLC

2

NB

ESC

Fibr

obla

sts

002040608

11214

2minusΔΔ

CT

(d)

LIN28

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

05

1

15

2minusΔΔ

CT

(e)

SALL4

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

05

1

15

2minusΔΔ

CT

(f)

PRDM14

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

2

4

6

8

10

2minusΔΔ

CT

(g)

DPPA3

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

5

10

15

20

2minusΔΔ

CT

(h)

Figure 5 Continued


DAZL

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

5

10

15

202minusΔΔ

CT

(i)

VASA

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

2

4

6

8

2minusΔΔ

CT

(j)

NOBOX

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts0123456

2minusΔΔ

CT

(k)

SCP3

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

002040608

112

2minusΔΔ

CT

(l)

OocyteDAPI OLCDAPI







(kDa)120100

80

60

50

50

40

40

VASA

(DD

X4)

Live

r

Stom

ach

Panc

reas

Skin

Testi

s

120573-a

ctin

Ova

rylowast

(a)

(b) (c)

(d) (e)

50120583m

(f)

50120583m

(g)

50120583m

(h)





4 Discussion



D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

NANOG

Fibr

obla

sts

002040608

11214

2minusΔΔ

CT

(a)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

OCT4

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(b)LIN28

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(c)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

SALL4

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(d)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

VASAFi

brob

lasts

0

02

04

06

08

1

12

2minusΔΔ

CT

(e)






















Acknowledgments


References





















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Component 1 (4270)

Com

pone

nt 2

(23

79

)

Neonatal ovary

Fibroblasts

OCCs

ES cells

150

100

50

0

minus50


LONBCjRONBCj

ESCcjes001P96

ESCcjes001P91


P4T1-C1

P4T2-C1



(a)

LOC100411838


ENSCJAT000000333

ENSCJAT000000532



LOC100391376



Ovary OCCsColor key

0 2Row Z-score

(b)

LOC100398751

LOC100405099LOC100387244


LOC100407886

LOC100402066

LOC100412217LOC100412217LOC100412217

KRT36GC

VCAM1VCAM1

LOC100393188





ENSCJAT000000054






0 2Row Z-score(c)

LOC100400589LOC100405014LOC100384946LOC100398697

LOC100408978


CDH1CDH1GYLTL1BCDH1

TDRD1

GDF3PDPN

LOC100386871

LOC100390443LOC100396000



ENSCJAT000000434


ENSCJAT000000159




0 2Row Z-score(d)

Figure 2 Continued







0 5 10 15 20 25

minuslog10(FDR)

(e)








002040608

11214

OCT4

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(a)

002040608

11214

NANOG

2minusΔΔ

CT

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

(b)

002040608

112

SALL4

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(c)

002040608

112

LIN28

2minusΔΔ

CT

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

(d)

0

05

1

15VASA

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(e)





002040608

11214

PRDM1

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(a)

002040608

11214

PRDM14

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(b)

002040608

112

DAZL

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(c)

002040608

112

DPPA3

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(d)

NOBOX

002040608

112

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(e)

SCP3

002040608

11214

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(f)





P23P10 P17 P20

(a)

(b)

OCT4

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

002040608

112

2minusΔΔ

CT

(c)

NANOGO

LC1

OLC

2

NB

ESC

Fibr

obla

sts

002040608

11214

2minusΔΔ

CT

(d)

LIN28

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

05

1

15

2minusΔΔ

CT

(e)

SALL4

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

05

1

15

2minusΔΔ

CT

(f)

PRDM14

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

2

4

6

8

10

2minusΔΔ

CT

(g)

DPPA3

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

5

10

15

20

2minusΔΔ

CT

(h)

Figure 5 Continued


DAZL

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

5

10

15

202minusΔΔ

CT

(i)

VASA

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

2

4

6

8

2minusΔΔ

CT

(j)

NOBOX

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts0123456

2minusΔΔ

CT

(k)

SCP3

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

002040608

112

2minusΔΔ

CT

(l)

OocyteDAPI OLCDAPI







(kDa)120100

80

60

50

50

40

40

VASA

(DD

X4)

Live

r

Stom

ach

Panc

reas

Skin

Testi

s

120573-a

ctin

Ova

rylowast

(a)

(b) (c)

(d) (e)

50120583m

(f)

50120583m

(g)

50120583m

(h)





4 Discussion



D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

NANOG

Fibr

obla

sts

002040608

11214

2minusΔΔ

CT

(a)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

OCT4

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(b)LIN28

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(c)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

SALL4

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(d)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

VASAFi

brob

lasts

0

02

04

06

08

1

12

2minusΔΔ

CT

(e)






















Acknowledgments


References





















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







0 5 10 15 20 25

minuslog10(FDR)

(e)








002040608

11214

OCT4

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(a)

002040608

11214

NANOG

2minusΔΔ

CT

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

(b)

002040608

112

SALL4

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(c)

002040608

112

LIN28

2minusΔΔ

CT

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

(d)

0

05

1

15VASA

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(e)





002040608

11214

PRDM1

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(a)

002040608

11214

PRDM14

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(b)

002040608

112

DAZL

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(c)

002040608

112

DPPA3

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(d)

NOBOX

002040608

112

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(e)

SCP3

002040608

11214

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(f)





P23P10 P17 P20

(a)

(b)

OCT4

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

002040608

112

2minusΔΔ

CT

(c)

NANOGO

LC1

OLC

2

NB

ESC

Fibr

obla

sts

002040608

11214

2minusΔΔ

CT

(d)

LIN28

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

05

1

15

2minusΔΔ

CT

(e)

SALL4

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

05

1

15

2minusΔΔ

CT

(f)

PRDM14

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

2

4

6

8

10

2minusΔΔ

CT

(g)

DPPA3

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

5

10

15

20

2minusΔΔ

CT

(h)

Figure 5 Continued


DAZL

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

5

10

15

202minusΔΔ

CT

(i)

VASA

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

2

4

6

8

2minusΔΔ

CT

(j)

NOBOX

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts0123456

2minusΔΔ

CT

(k)

SCP3

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

002040608

112

2minusΔΔ

CT

(l)

OocyteDAPI OLCDAPI







(kDa)120100

80

60

50

50

40

40

VASA

(DD

X4)

Live

r

Stom

ach

Panc

reas

Skin

Testi

s

120573-a

ctin

Ova

rylowast

(a)

(b) (c)

(d) (e)

50120583m

(f)

50120583m

(g)

50120583m

(h)





4 Discussion



D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

NANOG

Fibr

obla

sts

002040608

11214

2minusΔΔ

CT

(a)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

OCT4

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(b)LIN28

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(c)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

SALL4

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(d)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

VASAFi

brob

lasts

0

02

04

06

08

1

12

2minusΔΔ

CT

(e)






















Acknowledgments


References





















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


002040608

11214

OCT4

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(a)

002040608

11214

NANOG

2minusΔΔ

CT

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

(b)

002040608

112

SALL4

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(c)

002040608

112

LIN28

2minusΔΔ

CT

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

(d)

0

05

1

15VASA

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(e)





002040608

11214

PRDM1

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(a)

002040608

11214

PRDM14

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(b)

002040608

112

DAZL

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(c)

002040608

112

DPPA3

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(d)

NOBOX

002040608

112

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(e)

SCP3

002040608

11214

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(f)





P23P10 P17 P20

(a)

(b)

OCT4

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

002040608

112

2minusΔΔ

CT

(c)

NANOGO

LC1

OLC

2

NB

ESC

Fibr

obla

sts

002040608

11214

2minusΔΔ

CT

(d)

LIN28

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

05

1

15

2minusΔΔ

CT

(e)

SALL4

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

05

1

15

2minusΔΔ

CT

(f)

PRDM14

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

2

4

6

8

10

2minusΔΔ

CT

(g)

DPPA3

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

5

10

15

20

2minusΔΔ

CT

(h)

Figure 5 Continued


DAZL

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

5

10

15

202minusΔΔ

CT

(i)

VASA

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

2

4

6

8

2minusΔΔ

CT

(j)

NOBOX

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts0123456

2minusΔΔ

CT

(k)

SCP3

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

002040608

112

2minusΔΔ

CT

(l)

OocyteDAPI OLCDAPI







(kDa)120100

80

60

50

50

40

40

VASA

(DD

X4)

Live

r

Stom

ach

Panc

reas

Skin

Testi

s

120573-a

ctin

Ova

rylowast

(a)

(b) (c)

(d) (e)

50120583m

(f)

50120583m

(g)

50120583m

(h)





4 Discussion



D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

NANOG

Fibr

obla

sts

002040608

11214

2minusΔΔ

CT

(a)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

OCT4

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(b)LIN28

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(c)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

SALL4

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(d)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

VASAFi

brob

lasts

0

02

04

06

08

1

12

2minusΔΔ

CT

(e)






















Acknowledgments


References





















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


002040608

11214

PRDM1

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(a)

002040608

11214

PRDM14

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(b)

002040608

112

DAZL

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(c)

002040608

112

DPPA3

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(d)

NOBOX

002040608

112

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(e)

SCP3

002040608

11214

P1 P2 P3 P4 P5 P9 P11

P13

NB

ESC

Fibr

obla

sts

2minusΔΔ

CT

(f)





P23P10 P17 P20

(a)

(b)

OCT4

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

002040608

112

2minusΔΔ

CT

(c)

NANOGO

LC1

OLC

2

NB

ESC

Fibr

obla

sts

002040608

11214

2minusΔΔ

CT

(d)

LIN28

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

05

1

15

2minusΔΔ

CT

(e)

SALL4

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

05

1

15

2minusΔΔ

CT

(f)

PRDM14

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

2

4

6

8

10

2minusΔΔ

CT

(g)

DPPA3

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

5

10

15

20

2minusΔΔ

CT

(h)

Figure 5 Continued


DAZL

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

5

10

15

202minusΔΔ

CT

(i)

VASA

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

2

4

6

8

2minusΔΔ

CT

(j)

NOBOX

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts0123456

2minusΔΔ

CT

(k)

SCP3

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

002040608

112

2minusΔΔ

CT

(l)

OocyteDAPI OLCDAPI







(kDa)120100

80

60

50

50

40

40

VASA

(DD

X4)

Live

r

Stom

ach

Panc

reas

Skin

Testi

s

120573-a

ctin

Ova

rylowast

(a)

(b) (c)

(d) (e)

50120583m

(f)

50120583m

(g)

50120583m

(h)





4 Discussion



D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

NANOG

Fibr

obla

sts

002040608

11214

2minusΔΔ

CT

(a)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

OCT4

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(b)LIN28

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(c)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

SALL4

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(d)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

VASAFi

brob

lasts

0

02

04

06

08

1

12

2minusΔΔ

CT

(e)






















Acknowledgments


References





















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


P23P10 P17 P20

(a)

(b)

OCT4

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

002040608

112

2minusΔΔ

CT

(c)

NANOGO

LC1

OLC

2

NB

ESC

Fibr

obla

sts

002040608

11214

2minusΔΔ

CT

(d)

LIN28

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

05

1

15

2minusΔΔ

CT

(e)

SALL4

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

05

1

15

2minusΔΔ

CT

(f)

PRDM14

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

2

4

6

8

10

2minusΔΔ

CT

(g)

DPPA3

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

5

10

15

20

2minusΔΔ

CT

(h)

Figure 5 Continued


DAZL

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

5

10

15

202minusΔΔ

CT

(i)

VASA

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

2

4

6

8

2minusΔΔ

CT

(j)

NOBOX

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts0123456

2minusΔΔ

CT

(k)

SCP3

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

002040608

112

2minusΔΔ

CT

(l)

OocyteDAPI OLCDAPI







(kDa)120100

80

60

50

50

40

40

VASA

(DD

X4)

Live

r

Stom

ach

Panc

reas

Skin

Testi

s

120573-a

ctin

Ova

rylowast

(a)

(b) (c)

(d) (e)

50120583m

(f)

50120583m

(g)

50120583m

(h)





4 Discussion



D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

NANOG

Fibr

obla

sts

002040608

11214

2minusΔΔ

CT

(a)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

OCT4

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(b)LIN28

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(c)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

SALL4

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(d)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

VASAFi

brob

lasts

0

02

04

06

08

1

12

2minusΔΔ

CT

(e)






















Acknowledgments


References





















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


DAZL

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

5

10

15

202minusΔΔ

CT

(i)

VASA

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

0

2

4

6

8

2minusΔΔ

CT

(j)

NOBOX

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts0123456

2minusΔΔ

CT

(k)

SCP3

OLC

1

OLC

2

NB

ESC

Fibr

obla

sts

002040608

112

2minusΔΔ

CT

(l)

OocyteDAPI OLCDAPI







(kDa)120100

80

60

50

50

40

40

VASA

(DD

X4)

Live

r

Stom

ach

Panc

reas

Skin

Testi

s

120573-a

ctin

Ova

rylowast

(a)

(b) (c)

(d) (e)

50120583m

(f)

50120583m

(g)

50120583m

(h)





4 Discussion



D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

NANOG

Fibr

obla

sts

002040608

11214

2minusΔΔ

CT

(a)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

OCT4

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(b)LIN28

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(c)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

SALL4

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(d)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

VASAFi

brob

lasts

0

02

04

06

08

1

12

2minusΔΔ

CT

(e)






















Acknowledgments


References





















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(kDa)120100

80

60

50

50

40

40

VASA

(DD

X4)

Live

r

Stom

ach

Panc

reas

Skin

Testi

s

120573-a

ctin

Ova

rylowast

(a)

(b) (c)

(d) (e)

50120583m

(f)

50120583m

(g)

50120583m

(h)





4 Discussion



D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

NANOG

Fibr

obla

sts

002040608

11214

2minusΔΔ

CT

(a)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

OCT4

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(b)LIN28

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(c)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

SALL4

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(d)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

VASAFi

brob

lasts

0

02

04

06

08

1

12

2minusΔΔ

CT

(e)






















Acknowledgments


References





















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

NANOG

Fibr

obla

sts

002040608

11214

2minusΔΔ

CT

(a)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

OCT4

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(b)LIN28

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(c)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

SALL4

Fibr

obla

sts

0

02

04

06

08

1

12

2minusΔΔ

CT

(d)

D2W

1

D2W

2

D4W

1

D4W

2

D6W

1

D6W

2

D8W

1

D8W

2

NB

ESC

VASAFi

brob

lasts

0

02

04

06

08

1

12

2minusΔΔ

CT

(e)






















Acknowledgments


References





















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



















Acknowledgments


References





















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

long-term oocyte-like cell development in cultures derived … · 2015-11-11 · long-term...

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