1. Potential of Human amniotic fluid stem cells in recovery of ovarian function in mice Peyman Ghoraishizadeh (PHD candidate) Laboratory of Stem Cells and nano-regenerative medicine, University of Los Andes

2. Fetal stem cells shorten doubling time Less immunogenicity 3. Human amniotic fluid stem cells 4. Premature ovarian failure 5. Methodology 1. Amniotic fluid, follicular fluid and oocyte sample collection Amniocentesis oocyte pick up oocyte with fertilization failure 2. Preparation of follicular fluid 3.Human amniotic fluid cell culture and differentiation culture DMEM-f12, Bfgf, ES-FBS EB culture in media+5% Follicular fluid factor cocktail (Differentiation media) culture 6. Methodology 4. RNA Extraction and Real time PCR 5. Flow cytometry 6. Animal modules 7. Transfection and transplantation of human amniotic fluid cells 8. Immunohistochemistry analysis 9. Immunofluorescence staining 7. Results Theexpression ofstem and germ cell-specificgenes inundifferentiated human amniotic fluidcells(hAFCs).(A,B)QuantitativePCR wasusedtocomparestemcellandgermcellspecific geneexpressioninhAFCs obtainedfrom6independent samples,humanembryonic stemcells (hES)andhumanGV oocytes.Humanskin fibroblast cells (hSFC)wereusedasnegativecontrolsand18sRNA wasusedasan internalhousekeepinggene.Results shownrepresentmeanstandarddeviationfromthreeindependent experiments.(C) Immunofluorescenceanalysisofgermcell-specific genesinhumanGV oocytes.Scale bars=50m.(D)Immunofluorescenceanalysis of germcell-specific genesinundifferentiatedhAFCs.W hile hAFCs expressedOCT4,expressionwasnegativeforBLIMP1,DAZL,STELLA,ZPC andSCP3.Scale bars=50m. 8. Phase c o n t r a s t p h o t o m i c r o g r a p h s of human amniotic fluid cultured cells and s p o n t a n e o u s d i f f e r e n t i a t i o n of clone cells i n t o cells of three e m b r y o n i c germ layers. ( A , B) h A F C s formed clones (white a r r o w s ) and attached c e lls ( b la c k a r r o w s ) after 57 days in culture. ( C ) Clone c e ll s yielded embryoid bodies ( E B s ) ( b la c k a r r o w ) . (D) Scatter plots and bar graph for flow cytometry a n a ly s is of O C T 4 and CD117 expression in attached c e ll s ( h A F C ) and clones ( h A F C - c l o n e ) . The percentage of O c t 4 + / C D 1 1 7 + expressing c e ll s was higher ( 7 .8 % ) in clone t h a n attached c e lls ( 1 .8 1 % ; * * * P < 0 .0 0 1 ) ( * * P < 0 .0 1 , * P < 0 .0 5 ) . ( E ) E m b r y o id bodies spontaneously differentiated into c e l l s stained with S o x 1 7 , muscle actin and Nestin r e s p e c ti v e ly . M a g n if i c a t io n , 100x ( A ) , 200x( B , C , E ) . 9. Gene e x p r e s s i o n level of germ cell markers in e m b r y o i d bodies ( E B s ) derived from hAFCs and then cultured further for 1 o r 2 weeks (1 w, 2 w). ( A ) E B s cultured under standard d i f f e r e n t i a t i o n conditions, cultured in a d i f f e r e n t i a t i o n conditions with the addition of a germ cell maturation factor c o c k t a i l ( F A C ) or cultured in human f o l l i c u l a r f l u i d ( H F F ) - c o n t a i n i n g d i f f e r e n t i a t i o n medium. E B s not exposed t o differentiation conditions were used as negative controls for gene e x p r e s s i o n ; 18 s r R N A was used as an internal housekeeping gene. Data represents means S E of 3 independent e x p e r i m e n t s . ( B ) Immunofluorescence a n a l y s i s of c e l lu l ar and s u b c e ll u l a r protein expression for germ c e l l m a r k e r s in embryoid bodies ( E B s ) derived f r o m cultured human amniotic f l u i d c e l l s ( h A F C s ) . E B s were cultured f o r 1 week in basic d i f f e r e n t i a t i o n medium, germ c e l l maturation factor c o c k t a i l ( F A C ) - s u p p l e m e n t e d d i f f e r e n t ia t i o n m e d i u m or human f o l l i c u l a r f lu i d ( H F F ) - s u p p l e m e n t e d d i f f e r e n t i a t i o n medium S c al e b a r s = 100 m. 10. Grafted h A F C s infiltrate the c h e m i c a l l y - d a m a g e d murine ovarian tissue and r e s t o r e ovarian function. ( A ) Im m u n o h i s t o c h e m i s t r y for h u m a n - s p e c if ic antigens detecting grafted c e l l s r e s u l t in g f r o m transplantation of human am n i o t i c f l u i d c e l ls into the o v a r i e s of c h e m i c a l l y - s t e r il i z e d mice. (a) Sample for human n u c l e i an t ig e n - n e g a t i v e o v a r i a n section f o l l o w i n g h A F C transplantation. ( b , c) Human n u c l e i an t ig e n expression in r e c i p i e n t o v a r i e s 2 months after h A F C s t r ansp l a n t at i o n . (d) Sample for human f o l l i c l e stimulating hormone receptor ( F S H R ) - n e g a t i v e o v a r i a n s e c t io n f o l l o w i n g h A F C s t r ansp l a n t at i o n . ( K , L ) Human F S H R expression in r e c i p i e n t o v a r i e s 2 months after h A F C s t r a n s p l a n t at i o n . A r r o w s in d ic a t e p osit i v e expression of human n u c l e i or human F S H R in the o o c y t e , w h il e the other granulosa c e ll s served as negative c o n t r o l s . S c ale b a r s = 100 m. (B) Im m u n o h i s t o c h e m i s t r y for an t i- m u l le r i a n hormone ( A M H ) in o v a r ia n sections f r o m m i c e . (a) A M H i s expressed in the g r an u losa c e l l s of p r i m a r y , preantral and s m a l l a n t r a l f o l l i c l e s i n o v a r i e s f r o m control f e r t i le m i c e . (b) A M H expression disappears in the o v a r i e s of c h e m i c a l ly - s t e r il iz e d mice examined 2 months f o ll o w in g h A F C - f r e e medium t r a n s p l a n t at i o n . ( c , d) A M H expression reappears in o v a r i e s f r o m c h e m i c a l l y s t e r il iz e d mice w h e n examined at 2 months f o l l o w i n g h A F C t r a n s p l an t at i o n . S c a l e bars = 200 m 11. Conclusion hAFCs integrated into the ovaries of infertile mice and participated in oocyte regeneration. Thus ovarian function can be at least partially restored following hAFC transplantation. hAFCs can be alternative source for embryonic stem cells for treatment of POF and aging related infertility