ARTICLE
doi:10.1038/nature10637 Self-formation of functional adeno-hypophysis in three-dimensional culture
Hidetaka Suga1,2,3,Taisuke Kadoshima1,Maki Minaguchi1,Masatoshi Ohgushi2,Mika Soen1,Tokushige Nakano1,Nozomu Takata1, Takafumi W ataya1,Keiko Muguruma1,Hiroyuki Miyoshi4,Shigenobu Yonemura5,Yutaka Oiso3&Yoshiki Sasai1
The adenohypophysis(anterior pituitary)is a major centre for systemic hormones.At prent,no efficient stem-cell culture for its generation is available,partly becau of insufficient knowledge about how the pituitary primordium (Rathke’s pouch)is induced in the embryonic head ectoderm.Here we report efficient lf-formation of three-dimensional adenohypophysis tissues in an aggregate culture of mou embryonic stem(ES)cells.ES cells were stimulated to differentiate into non-neural head ectoderm and hypothalamic neuroectoderm in adjacent layers within the aggregate,and treated with hedgehog signalling.Self-organization of Rathke’s-pouch-like three-dimensional structures occurred at the interface of the two epithelia,as en in vivo,and various endocrine cells including corticotrophs and somatotrophs were subquently produced.The corticotrophs efficiently creted adrenocorticotropic hormone in respon to corticotrophin releasing
hormone and,when grafted in vivo,the cells rescued the systemic glucocorticoid level in hypopituitary mice.Thus,functional anterior pituitary tissue lf-forms in ES cell culture,recapitulating local tissue interactions.
The adenohypophysis,corresponding to the anterior and intermediate lobes of the pituitary gland,contains veral types of endocrine cells,which crete systemic hormones such as adenocorticotropic hormone(ACTH),GH,LH/FSH,TSH and prolactin1–3in respon to hypothalamus-derived releasing hormones.In contrast,the neuro-hypophysis(posterior pituitary)consists of the axons and cretory termini of hypothalamic vasopressin and oxytocin neurons.During early mammalian development,the adenohypophysis anlage origi-nates as a placode in the non-neural head ectoderm rostral and adja-cent to the anterior neural plate4,5(Supplementary Fig.1a)(hereafter, this part of non-neural ectoderm,expressing region-specific markers Pitx1and Pitx2,is simply referred to as rostral head ectoderm;this includes the oral ectoderm as a large part).The thickened placodal epithelium invaginates and subquently detaches from the oral ectoderm,becoming a hollowed epithelial vesicle,Rathke’s pouch (Fig.1a,b and Supplementary Fig.1b,c).The development of Rathke’s pouch depends on tissue interactions between the rostral head ectoderm and the rostral hypothalamus3–8.We sought to recapitulate this interactive microenvironment of the pituitary-formin
g morpho-genetic field using three-dimensional ES cell culture. Juxtaposing ectodermal layers formed in culture Mou ES cells can be induced to differentiate into rostral hypothalamic tissues when cultured as floating aggregates(typically3,000cells per aggregate)in a chemically defined medium lacking extrinsic growth factors(including no insulin;hereafter referred to as SFEBq culture9). However,quantitative polymera chain reaction(qPCR)analysis found only low expression levels of the rostral head ectoderm marker Pitx2in SFEBq-cultured ES cells(Fig.1c).Becau the embryonic anlage for rostral head ectoderm and hypothalamic neuroectoderm are adjacent(Supplementary Fig.1a–c),we wondered if a slight shift in positional information could promote the simultaneous genera-tion of both tissues within the same aggregate in SFEBq culture.We experimentally tested veral culture conditions known to affect early ectodermal patterning9–12,and found two that substantially incread Pitx2expression levels(Fig.1d and data not shown):a large cell-aggregation(LCA)culture(red;starting differentiation culture at 10,000cells per aggregate,instead of3,000;Supplementary Fig.2a) and bone morphogenetic protein4(BMP4)treatment(blue;
0.5m M,days0–7).BMP4plus LCA also incread Pitx2expression, but no clear additive effects were en over LCA alone(Supplementary Fig.2b).
In the LCA culture,both Pitx1/21rostral head ectoderm tissue and Rx1neural tissue formed continuous epithelia within the ES cell aggregates(Fig.1e–g;Supplementary Fig.2c and data not shown). The Rx1tissues were Chx102nestin1(Chx10also known as Vsx2; Supplementary Fig.2d),characteristic of rostral hypothalamic epithelia (retinal epithelia are Rx1but Chx101nestin2)9,13.Importantly,a layer of Pitx1/21epithelium reproducibly formed on the surface of the ES cell aggregates,adjacent and exterior to the Rx1neuroepithelia,which formed inner layers(Fig.1f,g).In contrast,treatment with exogenous BMP4,which is generally known to promote differentiation of non-neural ectoderm at the cost of neural differentiation10,12,inhibited the formation of hypothalamic tissues even at0.5nM,regardless of the aggregation size(Supplementary Fig.2e).Converly,treatment with the BMP antagonist dorsomorphin inhibited the generation of Pitx21 ectoderm,which was promoted in the LCA culture(Supplementary Fig.2f).Given that the expression of Bmp2and Bmp4(refs8,14)was incread under LCA conditions(Supplementary Fig.2g–i),the moderate elevation of endogenous BMP signals in the LCA culture, which was insufficient to inhibit hypothalamic differentiation,may have contributed to the spontaneous formation of rostral head ectoderm.
Self-formation of Rathke’s pouches in vitro
In the embryo,Rathke’s pouch develops on the midline of the rostral head ectoderm,which receives strong sonic hedgehog(Shh) signals3–7,15(Supplementary Fig.1d).Accordingly,treatment with the
生日快乐英文怎么说
1Neurogenesis and Organogenesis Group,RIKEN Center for Developmental Biology,Kobe650-0047,Japan.2Division of Human Stem Cell Technology,RIKEN Center for Developmental Biology,Kobe650-0047,Japan.3Department of Endocrinology and Diabetes,Graduate School of Medicine,Nagoya University,Nagoya,466-8550,Japan.4Subteam for Manipulation of Cell Fate,BioResource Center,RIKEN, Tsukuba305-0074,Japan.5Electron Microscopy Laboratory,RIKEN Center for Developmental Biology,Kobe650-0047,Japan.
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hedgehog (Hh)agonist N-methyl-N 9-(3-pyridinylbenzyl)-N 9-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane (smoothened agonist;SAG,hereafter)16(days 0–10)efficiently incread expression of the Rathke’s pouch marker 17,18Lim3(also called Lhx3)in LCA-SFEBq culture (Fig.1h).In a culture using venus-reporter ES cells for Lim3expression (Lim3venus/1;Supplemen-tary Fig.3a,b),SAG reproducibly induced Lim3::venus 1cells in .90%of LCA-SFEBq aggregates (25–30%of total cells on day 10;Fig.1i,j and Supplementary Fig.3c).Addition
al BMP4treatment (SAG 1BMP4under the LCA condition)had no additive effect (rather,an inhibitory effect;Supplementary Fig.3d)on Lim3::venus expression.
In SAG-treated LCA-SFEBq aggregates,the Lim3::venus 1cells formed multiple oval epithelial clusters (Fig.1k and Supplementary Movie 1;unlike Lim31neurons in the brain,they were Tuj12;Fig.1l).No concomitant induction of the mesodermal markers brachyury and Flk1(also known as Kdr)or the neural crest marker Foxd3was obrved in the aggregates (Supplementary Fig.4a–d).
The Lim3::venus 1tissues first appeared as thickened placode epithelium on the surface (,day 8),invaginated (days 9–10)and formed hollowed epithelial vesicles (days 10–11),as in embryonic
Rathke’s pouch development (Fig.2a–c and Supplementary Fig.4e).The Lim3::venus 1epithelium co-expresd Pitx1,Pitx2and E-cadherin,and was negative for neuroepithelial markers such as nestin,Rx and Nkx2.1or the surface ectoderm marker cytokeratin (Fig.2d–g and Supplementary Fig.4f–h),reminiscent of the marker profile of the pituitary primordium 1–3,19(Supplementary Fig.4i).The Lim3::venus 1epithelial vesicles were reproducibly located between the hypothalamic (Rx 1)and the rostral head ectodermal (Pitx11/Lim32)tissues,and the atypical protein kina C (aPKC)1apical surface of the Lim3::venus 1epithelial vesicle was inside along the apical–basal axis (
Supplementary Fig.4j,k),consistent with the topographical development of Rathke’s pouch in vivo .The pouch epithelia consisted of tall columnar epithelial cells with apical cilia and junctions (Fig.2h and Supplementary Fig.5),and the late-ont Rathke’s pouch marker 1,6,20islet1/2(Fig.2i)was expresd in a differ-entiating cell population located basally on day 13(e Fig.2h,bracket).On days 13–14,the long axis of the Lim31pouch typically reached a diameter of 150–200m m,comparable to that of early Rathke’s pouch in vivo .
Thus,in the prence of high Hh signals,the ES-cell-derived rostral head ectoderm adjacent to the hypothalamic neuroepithelium initiates
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s Figure 1|Adjacent formation of head ectoderm and hypothalamic epithelia in ES cell culture.a ,Schematic of pituitary development,sagittal view.A,anterior;D,dorsal;P,posterior;V,ventral.b ,In vivo expression of Pitx1and Rx on embryonic day 13(E13).c ,Little expression of Pitx2in SFEBq/CDM-cultured ES cells (qPCR;n 53experiments).d,day.d ,Pitx2expression in aggregates cultured under the LCA condition (red)or treated with 0.5nM BMP4(blue)(n 53).e ,f ,Immunostaining of day-6LCA aggregates for Pitx1(red),and Rx (green,f ).g ,Schematic of the result.h ,Induction of Lim3expression by SAG (qPCR;n 59).i ,FACS analysis of the Lim3::venus 1
population in day-10LCA aggregates with SAG treatment (green).Grey,day-1aggregate.j ,Lim3::venus expression in LCA aggregates with SAG treatment.k ,Clusters of Lim3::venus 1vesicles (arrowheads)in day-13SAG-treated LCA aggregates.Arrows,vesicles forming on the other side.l ,Lim31pouch is Pitx11but Tuj12.Scale bars,100m m (b ,e ,f ,k ,l );500m m (h ).The values shown on graphs reprent the NS,not significant.*,P ,0.05;**,P ,0.01;***,P ,0.001.
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Rathke’s pouch development in three-dimensional culture (Fig.2j).The interactions between the two juxtapod tissues emed to be critical,as Pitx1/21surface epithelia lacking Rx 1neural tissues in BMP4-treated aggregates (Fig.1d and Supplementary Fig.2e)did not exhibit visible pouch development,regardless of Hh signals (data not shown).In addition,surface ectoderm isolated alone from day-6aggregates exhibited low Lim3expression by qPCR when cultured until day 13,unless combined with hypothalamic neuroepithelium (Sup-plementary Fig.6a–c).
Fgf signals such as Fgf10are implicated in Rathke’s pouch develop-ment 6–8,21.Consistent with this idea,the Fgf receptor inhibitors SU5402and PD173074reduced the percentages of Lim3::venus 1cells in culture (Supplementary Fig.6d).Fgf10expression was gradually induced in the Rx 1tissues of the aggregate during days 7–10,and Fgf8expression was obrved in both Rx 1and Rx 2populations (Supplementary Fig.6e,f).phosho-Erk (pErk),which is indicative of Fgf signalling,predominantly accumulated in the Pitx11surface epithelia (Supplementary Fig.6g).This preferential pErk accumula-tion may be relevant to the localized expression of FgfR2b (Fgf10-specifi
c receptor gene 22)and FgfR4(receptor gene for Fgf8)in the Rx 2tissues (Supplementary Fig.6h,i).The obrvations indicate that endogenous Fgf signals also have a role in Lim3induction,at least in part,in this three-dimensional ES cell culture.
Generation of multiple endocrine lineages
During early pituitary development 23,Lim31pituitary progenitors become committed to veral hormone-type-specific lineages 1–6(Fig.3a;Supplementary Fig.7a for details).Among them,the ACTH-producing corticotroph lineage requires the transcription factor Tbx19(ref.24),the expression of which is inhibited by Notch signalling 25,26(Fig.3a).Consistent with this idea,whereas Tbx19expression in SAG-treated LCA-SFEBq aggregates was low,it was strongly incread by treatment with the Notch inhibitor (2S)-N-[N-(3,5-Difluorophenacetyl)-L -alanyl]-2-phenylglycine
tert-butyl ester (DAPT)(Fig.3b and Supplementary Fig.7b).Substantial numbers of large-sized ACTH 1cells reproducibly accumulated in the Tbx191domains of DAPT-treated pouch tissue (day 21;Fig.3d and Supplementary Fig.7c);ACTH 1cells comprid ,35%of N-cadherin 2non-neural cells (Fig.3c),which themlves reprented ,11%of total cells on day 21.The ACTH 1cells were negative for Ki67(not shown)and E-cadherin (Supplementary Fig.7d),unlike the early progenitors in the pouch
(for example,Supplementary Fig.4h).The ES-cell-derived ACTH 1cells were negative for the neuronal markers neurofilament and NSE (Fig.3e and Supplementary Fig.7e)and the melanotroph-lineage marker 24PC2(Fig.3f and Sup-plementary Fig.7f),consistent with the corticotroph nature of marker expression.
Lim3is esntial for early pituitary specification 17,18,27.Knockdown of Lim3(short hairpin RNA (shRNA)-bad;Supplementary Fig.7g)specifically reduced expression of Tbx19and Pomc (mou homologue of human ACTH ,hereafter referred to as the latter)in culture (Fig.3g;Supplementary Fig.7h for controls).Similarly,when Tbx192/2ES cells (Supplementary Fig.7i)were ud,ACTH expression was significantly reduced (Fig.3h).Thus,LCA-SFEBq culture with SAG treatment faithfully recapitulates in vivo corticotroph development,which is inhibited by Notch and driven by Lim3and Tbx19(Fig.3i).
In the developing pituitary,Tbx192progenitors give ri to Pit11intermediate precursors,which subquently differentiate into GH-,prolactin-and TSH-producing cell lineages 3–6,28(Fig.3a).Unlike Tbx19expression,Pit1expression in the SAG-treated aggregates was not incread by DAPT treatment (Fig.3j).In contrast,consistent with previous reports that canonical Wnt signalling promotes Pit1expression 28,its expression was specifically augmented by treatment with the Wnt agonist 6-bro
mo-3-[(3E)-1,3-dihydro-3-(hydroxyimino)-2H-indol-2-ylidene]-1,3-dihydro-(3Z)-2H-indol-2-one (BIO)(GSK3b inhibitor)on days 16–18(Fig.3j;e also Fig.3b,c).Further differ-entiation of the intermediate precursors into GH 1and prolactin 1cells was obrved (Fig.3k–n)when cultured in media containing glucocorticoid and oestradiol,respectively (Supplementary Fig.8a,b),which enhance the differentiation of the lineages 29,30.
Head menchyme has been suggested to have a promoting role in pituitary development 31.With this in mind,we treated LCA-SAG aggregates with conditioned medium of PA6stromal cells (derived from skull bone-marrow menchyme)(Supplementary Fig.8c).This treatment enabled the generation of LH 1and FSH 1cells (also some TSH 1cells;Fig.3o–r),which was otherwi very rare (data not shown).
The findings demonstrate the competence of ES-cell-derived adenohypophysis progenitors to generate multiple endocrine lineages in culture.
In vitro functionality as endocrine tissue
To investigatethe in vitro hormone-cretionability of adenohypophysis-like tissue generated in vitro ,we focud on corticotrophs,which were most efficiently generated in this three-dimensional ES cell
culture.Substantial ACTH relea from SAG 1DAPT-treated LCA aggregates was induced after a 10-min stimulation with corticotrophin releasing hormone (CRH)on day 21(Fig.4a,b and Supplementary Fig.9a),but not from tho without SAG treatment (Fig.4c).The aggregates treated with SAG but not with DAPT showed ACTH cretion upon
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Figure 2|Spontaneous generation of Rathke’s pouch-like vesicles in ES cell culture.a –c ,Morphogenesis of Lim31epithelia.d –g ,Immunostaining of day-12pouch vesicles and surrounding tissues for Pitx1(red,d –f ),Lim3(green,d ;white,e ;red,g ),pancytokeratin (green,e ),nestin (white,f )and Rx (green,f ,g )in ES cell culture.h ,Electron microscopy of the day-13pouch.
Delaminating cells on the basal side (bracket).i ,Islet11cells in the basal zone of the day-13pouch.j ,Schematic of in vitro generation of Rathke’s pouches.Scale bars,100m m (a –c ,e –g );50m m (d ,i );20m m (h ).
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stimulation,but significantly less than SAG 1DAPT-treated aggre-gates did (Fig.4c).
ACTH cretion from the pituitary is negatively regulated by the downstream hormone glucocorticoid
32(Fig.4d,int).Consistent with this in vivo regulation,the in vitro relea of ACTH by CRH was greatly suppresd by pre-treating with glucocorticoid (Fig.4d),but not with oestrogen (Supplementary Fig.9b).The findings demonstrate that the ES-cell-derived pituitary endocrine cells actively
crete ACTH and respond normally to both positive and negative regulators that work for endocrine homeostasis in vivo .
Functional rescue in hypopituitary mice
Lastly,we evaluated ACTH cretion in vivo using hypophyctomized mice 33(Fig.5a;e the reduction of blood ACTH levels in Sup-plementary Fig.9c,d).In mou,the pituitary is located at a tiny space in the skull ba,called the lla turcica 3–6.Becau its local circulatory
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Figure 3|Differentiation of ES-cell-derived pituitary progenitors into hormone-producing cell lineages.a ,Schematic of lineage-specific
differentiation.b ,Incread Tbx19expression on day 20by DAPT (days 18–19).BIO treatment (days 16–18).qPCR analysis (n 53experiments).
c ,Percentages of ACTH 1cells in non-neural (N-ca
d 2)cells on day 22(n 56).d –f ,Immunostaining of day-21SAG 1DAPT-treated aggregates for ACTH (
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g ,Attenuated Tbx19and ACTH expression by deoxycycline-induced Lim3knockdown (Dox;black columns)(n 56).
h ,Expression of Rx (day 7),Lim3(day 13)and ACTH (day 22)by qPCR analysis of LCA 1SAG 1DAPT-treated aggregates (n 53).Black columns,Tbx19knockout (Tbx19-KO)ES cells.
i ,Schematic of corticotroph
generation in vitro .j ,Pit1expression in day-26culture of LCA 1SAG
aggregates (n 53).k –o ,Non-corticotroph differentiation.k ,m ,Percentages of GH 1(k )and prolactin 1(PRL;m )cells in non-neural cells of day-33
LCA 1SAG aggregates.F&Ins,hydrocortisone 1insulin treatment;E2&Ins,oestradiol 1insulin treatment.
l ,n ,Immunostaining of day-33aggregates for GH (l )and prolactin (n )(green).o ,Percentages of non-neural cells producing LH,FSH and TSH in day-33aggregates cultured with PA6conditioned
medium during days 10–33.p –r ,LH,FSH and TSH immunostaining on day 33.Scale bars,20m m (d );50m m (e ,f );100m m (l ,n ,p –r ).The values shown on graphs reprent the *,P ,0.05;**,P ,0.01;***,P ,0.001.
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network including hypophyal portal veins is easily destroyed by hypophyctomy,we performed ectopic transplantation of aggregates under the kidney capsules in this study (Fig.5a;e grafted ACTH 1cells stained in red).
A week after transplantation,CRH loading induced a substantial elevation of blood ACTH levels in the mice grafted with SAG 1DAPT-treated aggregates (Fig.5b)but not in control hypophyctomized mice that had received aggregates without SAG/DAPT treatment (Fig.5c).Accordingly,the blood glucocorticoid (corticosterone)levels were incread upon CRH loading in the mice grafted with SAG
1DAPT-treated aggregates but not in controls (Fig.5d),indicating that ACTH from the graft sufficiently induced the downstream hormone.
Even without CRH loading,the basal levels of ACTH were higher in the mice that received SAG 1DAPT-treated aggregates than in controls (Fig.5e).Importantly,the corticosterone levels were also incread (Fig.5f),suggesting that this partial recovery of blood ACTH has a moderate but biologically significant effect (note that the median effec-tive do (ED 50)of the ACTH receptor MC2R for glucocorticoid pro-duction is around 9pg ml 21;ref.34).In accordance with this idea,the hypophyctomized mice receiving SAG 1DAPT-treated aggregates showed higher spontaneous locomotor activity in their home cages than controls (infrared-monitored 24-h tracking in Fig.5g;also examined by the running wheel test;Fig.5h and Supplementary Movie 2;Supplementary Fig.9e,f for controls),and survived signifi-cantly longer (P ,0.01,Kaplan–Meyer method;Fig.5i).
Conclusion and perspective
Although the adenohypophysis is relatively small in size,it is an inde-pendent endocrine organ with indispensable systemic functions.Using a synthetic approach at the multi-cellular level,we demonstrated that a functional organ bud,the development of which requires intricate
console
tissue interactions,can be reproducibly generated by lf-formation when the spatial arrangement of a few interacting tissues is recon-stituted in three-dimensional culture.In terms of mechanistic com-plexity,this idea reprents a further conceptual advance beyond our recent report of optic-cup lf-organization,which sufficiently occurs within retinal epithelium alone 13.
Hypopituitarism is a major category of endocrinological disorders,including empty lla syndrome,Sheehan syndrome,hypophyal apoplexy and post-operative pituitary damage 1,2.In addition,in-sufficiencies of specific hormones (for example,GH insufficiency in infants and adults)also cau various systemic problems.The successful generation of pituitary endocrine cells shown here opens new avenues for the application of pluripotent stem cells to treat non-diabetic endocrine dias,which have so far received relatively little attention in regenerative medicine.A future challenge will be ortho-topic transplantation of adenohypophysis derived from human ES/induced pluripotent stem cells into the lla of a larger mammal,together with local reconstitution of vascular
networks.
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Figure 4|CRH-induced ACTH relea from ES-cell-derived pituitary
tissues.a ,CRH efficiently induced ACTH cretion (n 54experiments).b ,ACTH cretion at different CRH dos (n 54).c ,ACTH cretion from aggregates cultured in different conditions (n 53).d ,Pre-treatment with hydrocortisone (n 53)suppresd the CRH-stimulated ACTH cretion from aggregates.The values shown on graphs reprent the *,P ,0.05;**,P ,0.01;***,P ,0.001.
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CRH loading test basal levels Figure 5|Incread systemic ACTH and glucocorticoid levels by grafting ES-cell-derived pituitary tissues.a ,Schematic of transplantation procedures.Int,ACTH 1cells (red)in grafted tissue (labelled with GFP;post-transplantation day 7)under the renal capsule.b ,Blood ACTH levels in grafted mice with (right)or without (left)CRH loading (n 53).S 2D 2,aggregates without SAG or DAPT treatment;S 1D 1;aggregates with SAG 1DAPT treatment.c ,d ,Blood ACTH (c )and corticosterone (d )levels upon CRH loading (sampled two hours after loading;7days after transplantation;n 53).Sham,subcapsular saline injection.e ,f ,Basal blood levels of ACTH (e )and corticosterone (f )7days after transplantation (n 53).No CRH loading.Blood sampling at 20:00in the evening.g ,Spontaneous locomotor activity (moving distance)in the home cage measured for 24h by
the infrared monitoring system.WT,wild type.Control (n 58)versus S 1D 1(n 57),P ,0.05,Bonferroni’s test.Wild type (no hypophyctomy),n 54.h ,Locomotor
measured by running wheels.Control (n 59)versus S 1D 1(n 57),P ,0.01.Wild type,n 54.i ,Improved survival of transplanted hypopituitary mice.S 2D 2,n 512;S 1D 1,n 514.Sham,n 512.The values shown on graphs reprent the *,P ,0.05;**,P ,0.01;***,P ,0.001.Scale bar,100m m (a ).
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METHODS SUMMARY
The SFEBq culture was performed as described previously except that cells were aggregated at10,000cells per aggregate in96-well plates and cultured in growth-factor-free chemically defined medium(gfCDM)medium supplemented with 400nM SAG on days0–10unless otherwi stated.For endocrine cell differenti-ation,culture was continued under40%O2.Surgical hypophyctomy was per-formed by the transaural approach33.
Full Methods and any associated references are available in the online version of the paper at www.
荒谬的英文
Received26July;accepted14October2011.
Published online9November2011.
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Supplementary Information is linked to the online version of the paper at
/nature.
Acknowledgements We are grateful to H.Enomoto,R.Ladher and M.Eiraku for invaluable comments,to K.Misaki for electron microscopy analysis,and to members of the Y.S.laboratory for discussion.This work was supported by grants-in-aid from Ministry of Education,Culture,Sports,Science and Technology(Y.S.,Y.O.),the Knowledge Cluster Initiative at Kobe,and the Leading Project for Realization of Regenerative Medicine(Y.S.).
Author Contributions H.S.and Y.S.designed the project and wrote the manuscript.
H.S.,T.K.,M.O.and M.M.performed the experiments with the technical help and advice of T.N.,N.T.,M.S.,K.M.,H.M.,S.Y.and T.W.,and Y.O.provided critical advice on the rearch strategy and design.
Author Information Reprints and permissions information is available at
/reprints.The authors declare no competing financial interests. Readers are welcome to comment on the online version of this article at私语的意思
/nature.Correspondence and requests for materials should be addresd to Y.S.(yo
shikisasai@cdb.riken.jp).wolf什么意思
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