Human amniotic fluid cells (hAFCs) may differentiate into multiple cell lineages and thus have a great potential to become a donor cell source for regenerative medicine. The ability of hAFCs to differentiate into germ cell and oocyte-like cells has been previously documented. Herein we report the potential use of hAFCs to help restore follicles in clinical condition involving premature ovarian failure.

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

6. 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

7. 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

8. 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 shownrepresentmean±standarddeviationfromthreeindependent experiments.(C)
Immunofluorescenceanalysisofgermcell-specific genesinhumanGV oocytes.Scale bars=50μm.(D)Immunofluorescenceanalysis of
germcell-specific genesinundifferentiatedhAFCs.W hile hAFCs expressedOCT4,expressionwasnegativeforBLIMP1,DAZL,STELLA,ZPC
andSCP3.Scale bars=50μm.

9. 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 5–7 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 ) .

10. 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 μm.

12. 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 μm. (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 μ m

13. 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