' • , % ,

E L S E V I E R Mechanisms of Development 45 (1994) 163-171

Maintenance of the pluripotential phenotype of embryonic stem cells through direct activation of gpl30 signalling pathways

Kanji Yoshida ~, Ian Chambers b, Jennifer Nichols h, Austin Smith ~, Mikiyoshi Saito ", Kiyoshi Yasukawa ", Mohammed Shoyab d, Tetsuya Taga ~"*, Tadamitsu Kishimoto e

" Institute.for Moh'cular and Celhdar Biolo,gy, Osaka Unicersity. 1-3 Yamada-oka, Suita, Osaka 505, Japan, t, AFR(" Centre tbr (;em~me Research, Unicersity q f Edinburgh, Kings Buildings, West Mains Road, EH9 3JQ, UK, ' Bioteclmolo,w Research Center. TOSOH Colporation, 2743-I

Hayakawa, Ayase, Kanagawa 252, Japan, d Fred Hutchinson Cancer Research Center, 1124 Columbia Street, EP31ON, Seattle. WA 08104, USA, " Department ~)[Medicine I11. Osaka Unicetwio' Medical School, 2-2 Yamada-oka, Suita, Osaka 505, Japan

(Received 11 October 1993: revised 8 November 1993; accepted 9 November 1993)

Abs t rac t

Propagation of the undifferentiated pluripotential phcnotype of cmbryonic stem (ES) cells is depcndent on the cytokinc differentiation inhibiting activity/leukemia inhibitory factor (DIA/LIF). Thc DIA/LIF receptor complex is a hetcrodimer of D I A / L I F receptor (DIA/LIF-R) and gpl30. The latter is also a component of the interleukin-6 (IL-6) receptor complex. We report that a combination of IL-6 and soluble IL-6 receptor (sIL-6R), which can induce homodimerisation of gpl30 and activation of signalling processes, sustains self-renewal of pluripotential ES cells. Our findings indicate that the IL-6/sIL-6R complex acts on ES cells through gpl30 alone, bypassing DIA/LIF-R, and therefore implicate gpl30 as the key component in the signalling pathway responsible for stem cell renewal.

Key words: Differentiation inhibiting activity; Leukemia inhibitory factor: Ciliary neurotrophic factor; Oncostatin M; Signal transduction; Self-renewal; Interleukin-6

I. Introduction

Murine embryonic stem (ES) cells are established stem cell lines derived from the inner cell mass of the preimplantation embryo (Evans and Kaufman, 1981; Martin, 1981). The cytokine D I A / L I F has a key role in the maintenance of the pluripotential phenotype of ES cells in vitro (reviewed by Smith, 1992). In the absence of a source of D I A / L I F , the stem cells differ- entiate and cannot be propagated. By contrast, ES cells cultured in the presence of D I A / L I F maintain their proliferative capacity, retain charactcristic stem cell morphology and express stem cell marker proteins such as alkaline phosphatase and stage-specific embryonic antigen-I (SSEA-1) (Williams et al., 1988; Smith et al., 1988). Most significantly, ES cells expanded in the presence of D I A / L I F remain pluripotential and com- petent to produce chimaeras when reintroduced into

* Corresponding author. Tel.: + 81-6-877 5802. Fax: + 81-6-877 1955.

Elsevier Science Ireland Ltd. SSDI 0 9 2 5 - 4 7 7 3 ( 9 3 ) E 0 0 7 0 - O

mouse blastocysts (Nichols et al., 1990; Pease et al., 199(1).

The mechanism by which ES cell self-renewal is achieved is poorly understood, but it is apparent that DlA/LlF-s igna l l ing is initiated by binding to a high affinity cell surface receptor complex (Williams et at., 1988; Smith et al., 1988). This is composed of a D I A / LIF-specific subunit ( D I A / L I F - R ) and the glycopro- tein gpl30 (Gearing et al., 1991, 1992; lp et al., 1992; Liu et al., 1992; Taga et al., 1992; Davis et al., 1993). Both components are members of the cytokine recep- tor family (Cosman et al., 1990; Bazan, 1990) and possess cytoplasmic domains which have been impli- cated in signal transduction (Hibi et al., 1990: Bau- mann et al., 1992). gpl30 was originally identified as a signal-transducing subunit of the IL-6 receptor com- plex (Taga et al., 1989: Hibi et al., 1990). gpl30 is also an essential component of functional receptors for oncostatin M (OSM), ciliary neurotrophic factor (CNTF) and interleukin-l l (IL,-ll) (Ip et al., 1992; Taga and Kishimoto, 1992; Taga et al., 1992: Kishimoto et al., 1992; Davis et al., 1993: Yin et al., 1993). An

164 K. )TMfida vl al. / ,,th,{ t, amsm v qt l Jes'clulmWm 4,5 1 Y,*?4 ; 103 171

cxtraccllular soluble lorm of the IL-6 receptor (slL-6R) can associate, when occupied by IL-6, with membrane- anchored gp 130 to actiwitc celluhir signalling processes (Taga ctal . , 1989; Yasukawa el al.. 1990). This suggests thal the intcractioll between IL-6R and gpl3() occurs cxtraccllularly and that the cytophismic region of IL-BR is dispensable for signalling. The addition of slL-6R can therefore (i) potentiate the actions of IL-6 on cells ahcady responsive to IL-6 or ( i i) confer Ik-6 respon- siveness to cells which lack t ransmembrane IL-6R but express gpl30. Recent observations have indicated that II,-(>stinluhltion induces formation of gpl3() homod- hncrs whereas DIA/LIF-s t imula t ion restllts in the het- crodimcrization of gpl30 and D I A / L I F - R (Murakami ct al., lC193: l)avis et al., 1993). I:ormation of cither gpl30-gpl3(I homodimers or g p l 3 I I - D I A / L I F - R het- crodinlcrs is associated with protein kinasc actiw~tion and is considered to hc the first step in the signalling processes of IL-6 and D I A / L I F , respectively.

lJascd on these obscrwltions, wc have dctermincd that [~S cells arc responsive to CNTF and OSM. Fur- ther to this, hccause KS cells arc IL-6R / g p l 3 0 ' (Saito cl a]., 1992) and thus unresponsive to IL-h alone, we have examined the activity of a combination of IL-6 plus slL-6R. We have compared biological and bio- chemical responses of ES cells to the I L - 6 / s l L - 6 R complex with those to D I A / l , l F and demonstrate that they have cquiwllent effects on KS cell self-renewal. Wc prcscnl evidence that whereas D I A / l J F - s i g n a l i n g hwolves hoth receptor components, the signal from the IL-(~/slI,-6R complex is transduced through gpl3() in- dcpcndcntly of I ) I A / I , I F - R .

2. Results

2. 1. t;'ormalion o / tmd(['ferenliated E S cell colonies i n the

prc.vence o[ 11=6 ph~s x l l , -6R

KS cells cultured in the presence of D I A / L I F form colonies with high Icvels of alkaline phosphatasc, a

A

B

C

D

A,C 4000 2000 1000 500 250 125

B,D 62 31 16 8 0 125 *

A

B

C

D

Concentration

~... 7r ,\,~-:~" ~ '~ ~ .'~' l,'. :". : , i ~ v . ~ , I: t!.. )j ,,, ~:~" '~ *'* -,"- " • -¢" x" .~';

100 10 1 0.1

Concentration

,, i

• • D S

Fig. I. Formation ~flcolonies and enhanced D N A s vnlhesis in KS ccl] cul lurc in the i)rc'~¢ncu of D IA / I _ IF , ( 'NTF, OSM and l l .-6-sl l .-6R. (Top and middle pancN) Formal ion o1" ES cull colonies, with alkaline I~hoslqlalas¢ aclJvil~. (i) [:.S ceils cultured in lhc prcscncc of ral ( ' N T t - ( r o ' , ~ A and lJ) or routine D I A / I H . (rows (" and D). ('onccntralions arc in pg/ml and the asterisk denote.', the addition el

nculraIizhL~ rahhil anti DIA/LIF anliscra: (ii) ES ceils cuIlurcd in

the presence of human rccombinanl factors DIA/LIF (row A), ]L-6 (rov, B). If. n plus ~,IL-nR (iov~. C). or OSM (row D). OSM is

expressed ;i~, ng/ml : I)IA/LIF and 11.-6 arc expressed as uni ts /ni l • I() :. Wells cultured with medium (*)or sII.-6R (~:*)ahmc arc

shov, n in Ihc hox. (Bol tom panel) D N A synlhcsis in KS cells. [)3 cells v, crc cultured lot 2 days in %-well plalcs in the presence of various

factor,, indicated in lhc figure and lhcn puls¢-hlhelcd wilh

[ ~II]lhymidhlc for 17 h. Data rcprc~,cI1t the incorporaled radioactiv

ib.

E t ~

o

F- " 0

E £

3 0 0 0 0

20000

10000

LIF IL-6+ slL-6R

OM IL-6 slL-6R medium

K. Yoshida ct al. / Mechaltism,~ ~>f" Del 'elopmen t 45 ( 1 ~)94 ) 163-17 l 165

m a r k e r of the u n d i f f e r e n t i a t e d p h e n o t y p e ( P e a s e et al.,

1990). ES cel ls w e r e c u l t u r e d in the p r e s e n c e o f C N T F

or O S M to inves t iga te w h e t h e r t h e s e cu l t u r e c o n d i t i o n s

s u p p o r t e d the g rowth of co lon i e s exp re s s ing this enzy-

ma t i c ma rke r . As shown in Fig. 1A, the add i t i on of

t he se cy tok ines to the cu l t u r e m e d i u m r e s u l t e d in the

d o s e - d e p e n d e n t a p p e a r a n c e of co lon i e s s ta in ing in-

t ense ly for a lka l ine p h o s p h a t a s e . T h e c o n c e n t r a t i o n s of

D I A / L 1 F and C N T F r e q u i r e d to el ici t a s imi la r re-

sponse in this assay a re e s sen t i a l ly ind i s t ingu i shab le .

Th i s e f fec t on ES cell d i f f e r e n t i a t i o n is spec i f ic and is

no t o b s e r v e d wi th a b r o a d r a n g e o f o t h e r cy tok ines

(Smi th and H o o p e r , 1987; and u n p u b l i s h e d data) .

H o w e v e r , in t he l ight o f r e c e n t f ind ings on the ut i l isa-

t ion of bo th D I A / L I F - R and g p l 3 0 as c o m m o n c o m -

p o n e n t s in the r e c e p t o r c o m p l e x e s for D I A / L I F ,

E

Q)

t13 G) n-

F luorescence intensity

Fig. 2. Expression of SSEA-1 antigen in ES cells cultured in the presence of DIA/LIE. 1L-6/slL-fiR and OSM. (Top) Flowcytometric analysis of SSEA-I expression. El4 ES cells were cultured in the presence of DIA/LIF (A). IL-6 together with slL-6R (B), OSM (C), IL-6 (D), slL-6R (E), or medium alone (F). Ceils were harvested and stained with anIi-SSEA-1 antibody and fluorescein-conjugated anti-mouse lgM. Solid profiles represent the staining detected by FACScan. The open profile in each panel indicates the SSEA-I staining of ES cells cultured in medium alone. A staining profile of the DIA/LIF-maintained ES cells with fluorescem-conjugated anti-mouse IgM antibody alone was superimposable to that shown in (F). (Bottom left) SSEA-I expression of colonies of ES cells. El4 ES cells cultured under the same set of six different conditions (A F) as in (A) were analyzed by fluorescent microscopy after fixafi,,~n and subsequent staining with anti-SSEA-I antibody and fluorescein-conjugatcd anti-mouse IgM. Magnification, × 100. (Bottom right) Morphology of ES cells. El4 IES cells cultured under the same set of six different conditions (A-F) as in (A) were analyzed by phase contrast microscopy after fixation. Magnification, × 400. Representative colonies of ES cells from the culture with DIA/LIF, IL-6-slL-6R, ,or OSM in (B) and (C) are shown.

I f)n K. ) } M t i d a cl aL/,~h,cttaslLvms' o/l&,t 'Ch>l>m(,ttt 45 ( 19747 1(~.7 - I ~1

CNTF and OSM (reviewed by Kishimoto el al,, 1992) it is perhaps not surprising thai the latter two factors havc similar activity to D I A / L I F on ES cells. More significant, though, is the finding thai the complex of IL-6 plus slL-6R also supported the formation of ES cell colonies possessing high levels of alkaline phos- phatase.

The differentiation of ES cells is also accompanied by a reduction in growth ratc. Therelorc, the effect of the I L - 6 / s l L - 6 R complex as well as of OSM lind D I A / L I F on KS ceil growth was examined by measur- ing the incorporation of [~H]thymidine into the cells maintained with these factors. As shown in Fig. I B, ES cells cultured in thc presence of D I A / L I F , the IL-6 / slL-6R complex, or OSM showed simihir enhance- mcnis of DNA synthesis. ES ceils maintained in the prescncc of D I A / L I F form compact colonies of small cells in contrast to the large, fhittcned, difTercntiated cells obtained in its absence (Smith el al., 1988). ES cells maintained m the presence of CNTF (not shown), OSM or the 1L-6/slL-6R complex exhibited this char- acteristic ES cell morphology, which was indistinguish- able from that observed with D I A / L I F (Fig. 2C).

The phcnotypc of ES cells cultured under the differ- ent conditions was compared by examining additional characteristics of undifferentiated ES cells. Expression of SSEA-I, a marker of undifferentiated mouse ES cells (Solter and Knowlcs, 1978; Martin, 1981; Smith el al., 1988), was examined by flowcytometric and im- munochemical analysis using a monochmal antibody against SSEA-I and fluorescein-conjugated second an- iibody. Flowcytometric profiles shown in Fig. 2A indi- cated that ES cells cultured with mcditnn supple- mented with either D I A / L I F or thc I L - 6 / s l L - 6 R complcx cxprcsscd substantial lcvels of SSEA-I. In contrast, SSEA-I was lost during culture without these supplements. In the experiment shown in Fig. 213, ES cell colonies were fixed lind stained for in situ fluores- cent microscopic analysis of SSEA-I expression. Cells in colonies formed in the presence of IL-6 plus slL-6R

"1 able I Produclhm of cinmaclas ITom ES cells cloned and propagated in the pi-csencc o f ihc 1L f~ /s lL -6R complex

('lone 13histocysls N(). el No. of injccicd progeny ~' chimaeras

(GR2.1 115 35 7 ( ( i 1),2.2 39 19 I 2 ( ( ; RS.4 19 ~ n

( 'GR2 and ( 'GR8 arc cuploid ES cell lines established from strain 129 ( c ' s ' / c 's') embryos by published procedures (Nichols el al., 1990). Sul)cioncs were isohllcd and cxpanded in the prcsencc o1" IL 6 phls sll.-6R for 2(1-3(I generations, then microiniccted into (57B1./() bhlsh)cysts. ( 'himacrism amongst resultant offspring was revealed by lhc plCS¢llCC ,,11 sandy co:.it colotlr piglllelliaii,.HI. ~' The nunlb¢ls o f SUl~,iving progcn~ wcrc reduced due to a high raic oJ llCOllilla] (.'anllibaJislll.

Tahlc 2 Pcrceillagc ['IS cell cl)rllrihuli,.)n h) di l fc rcn l tisstlcs in acitlll chi nl{iCl{ik

f:.S ( ' lone I. ivcr Spleen l l ca r i l .ung "l'cslcs Kidney Muscle Brain

( ' ( i R 2 . 1 3() 3(i 4o ~l) 4o 4(i su ~() ('(iR2.2 () 4{) >() 21) 30 4() :ql I()

T\~.o lllal¢ C]lilllaCl'ilb ~,%cI't.' SaCliJic.'cd {11 -I 111()111hs o ] ;l~t: ;llld lis<,tlCS processed lk)i GPI aimi)sis, lhc rclali~c p lopo r l i o l> ol q i ; i in 12(J 115; ccl l-dcri~cd ( iP I A,,\ Io ( ' 5 7 l / I f~ hosl derived ( l l ) l l i b arc e i tcn h*i Ihc ViiliOtlS Ii>,StlCS {llltl Ol'g~lll,, c'X~llllillcd.

showed bright uniform SSEA-1 staining. ES cells cul- tured without supplementation or supplemented with IL-6 alone or s lL-6R ah)nc formed a fhil cell hiycr lacking appreciable SSEA-1 cxprcssi()n. The platirlg efficiency of ES cells al clonal density ill lilt presence of IL-6 plus slL-6R was comparable to ttmt obtained in the presence of DIA/1 ,1F (15-25<7) and likewise the great majority of colonies contained slem cells. This indicates that the cffccl of the IIM~-slI,-6R complex is not duc to cell selection. Moreover, reproducible ob- servations have been m:.ide with several indcpcndcnl ES cell lines.

2. 2. (;eneratio#t o/" chimaura,s fiom I:S cdLs l)r,l)agaled with IL-6 plus slL-6R

The essential test for the normal developmental potential of ES cells is examination (/l the ability of clonal lines to form chimaeras when reintroduced to mouse blastoeysts (Bradley cl al., 1984). Therefore, ES ceils were cloned from single cells in lhc presence of IL-6 (200() u n i t s / e l ) plus sIl.-6R (2 # g / e l ) and b[as- tocyst injections were carried ()tit after 20-30 genera- tions. Three indcpendent clones were used lin injec- tions. Injected bhistocysts were transferred into pscu- dopregnant females and chimeric offspring were pro- duced (Table 1). The proportion of chimaeras and thc degrees of coat colour chimaerism obtained wcrc coin- parable to those observed with subcloncs isohltcd in parallel experiments in tile presence of I ) I A / L I E All the animals were hcalthy with no indications of ally abnormalities. The organs of two lypieal chimaeras were analyzed for the prcsencc o1' ES cell-derived ghicosc phosphate isomcrase (GPI) isocnzymc. As summarized in Table 2, the analysis confirmed that ES cells propagated in the presence of II,-6 plus s l l J6R contributcd to the normal developmcni of all tissues investigated. Germ-line transmission ()1: the f~S cell genotypc has not been obtained from lhe ( 'GR2 chi- maeras. However. since thcse experiments were inili- alcd it has bcconlc apparent thai the original gcrnl-l inc conlpctencc of tile ( '(}R2 ccll line has been lost and subclones isohtted in the presence of I ) I A / L I F also fail to givc germ-linc transfer. Thc experiment has therclkwc been repealed using the ( 'GR8 cell line

K. Yoshida et ul. / Mechanisms of Deteh)pment 45 (1994) 163-171 167

which gives more consistent germ-line transmission. New chimaeras have been generated (Table 1) and these animals will be test-bred on reaching sexual maturity.

2.3. Biochemical analysis of ES cells stimulated with IL-6 plus slL-6R

Low levels of D I A / L I F transcripts can be detected in undifferentiated ES cells by RNase protection assay (Rathjen et al., 1990a). The possibility that the IL-6/ slL-6R complex might upregulate the level of this transcript and thus may induce an autocrine effect on stem cell maintenance was examined. As shown in Fig. 3A, the undifferentiated DIA/LIF-main ta ined ES cells expressed low levels of D I A / L I F mRNA and this remained unchanged when the cells were cultured in medium containing IL-6 plus slL-6R. Therefore the effects of the IL-6 /s lL-6R complex are not mediated via induction of D I A l LIF, but are likely to result from direct stimulation of a stem cell signalling pathway.

Induction of the immediate early response gene TISI1 (Varnum et al., 1991) has previously been demonstrated in myeloid cell lines stimulated with Ik-6 (Nakajima and Wall, 1991) and in neuronal cell lines stimulated with D I A / L 1 F (Ip et al., 1992). TISI1 mRNA was induced by D I A / L I F in ES cells with maximal steady state levels apparent approximately 40 rain following D I A / L I F addition (Fig. 3B and data not shown). Moreover, T IS l l mRNA was induced in ES cells treated with a combination of IL-6 and sIL-6R, but not with IL-6 or slL-6R alone, nor in a mock-in- duced control. TISI1 may therefore be an indicator of subsequent ES cell self-renewal and its induction by both D I A / L I F and IL-6 / s lL-6R in ES cells suggests that these agents may trigger a common signal trans- duction cascade.

O

P t R ,", '~ l 2

D

,ii~ii~ii )

,.o ,.o

- - T I S 1 1

2.4. The actions of IL-O /slL-6R on ES cells are medi- ated through gpl30 and bypass DIA /LIF-R

We next examined whether the IL-6 / s lL-6R com- plex actually associates with gpl30 in ES cells. [35S]methionine-labeled cells were stimulated with a combination of IL-6 and sIL-6R and solubilized with NP40. The lysates were subjected to immunoprecipita- tion with an anti-IL-6R monoclonal antibody. As shown in Fig. 4A, protein with a MW of about 130 kD was detected in the immunoprecipitates, consistent with the expected interaction of 1L-6-occupied slL-6R with gpl30. In the same autoradiogram, however, there was no indication of a higher MW band which could corre- spond to co-precipitation of D I A / L I F - R (Gearing et al., 1991; Ip et a[., 1992; Taga et al,, 1992; see also Fig. 4B).

Based on recent reports showing rapid tyrosine-

2 8 S r R N A

1 8 S r R N A

Fig. 3. Unchanged expression of D1A/LIF and upregulation of TISII transcripts following stimulation of ES cells with 1L-6 plus slL-OR. (Top) D1A/LIF expression. CGR2 ES cells were cultured tot 26 h in media containing DIA/LIF (lane 1) or 1L-6 plus slL-6R (lane 2) and analyzed for D1A/L1F Iranscripts by RNase protection assay. Lane P contains undigested DIA/LIF probe. D and M indi- cate the positions of the alternative transcripts for diffusible and matrix-associated DIA/LIF (Rathjen ctal., lt)t~llb) and G marks the glyceraldehyde 3-phosphate dehydrogcnasc (GAPDIt) loading con- trol protection. (Bonom) Induction of T1Sll mRNA by DIA/LIF and the IL-6-sIL-6R complex. RNA from CPI ES cells treated as indicated or mock stimulated (0) was prepared and subjected to Northern analysis. The lower panel shows Ihc ethidium bromide staining of the gel prior to transfer.

1(~8 Ix'. )})~/|ida ul al. /' .,lh'chani.sn|.s ~1 lh'l'(,lOltllt~'ltl 45 ( ] ()~)4) 1( "~ 17/

.A B

slL-6R + + 1 IL-6 - + Mr(k)

200 - - 200 -

Mr(k) " 'i " a ' ~ ~ J f k * x : ~

, !

2 3

I gp130

Fig. J. Association ol gpl30 with the IL-flIsll,-bR colnplcx in KS cells and lyrosmc-phosphorylation of anli-gp 130 immunoprecipilates. (A) [~'S]mclhioninc labeled El4 KS cclb, ,,,,ctc stimulated by the I'actols indicalcd iu lhc figure. NP4(I lysatcs from the cells wcrc immunoprc cipilalcd wilh anti 1L-nR monoclonal antibody and analyzed by SI)S-PA(;IZ and autoradiography. (B) El4 KS ceils wcie slimulated with I ) I A / L I F (lane I). IL-fl-slL f~R (lane 2), or medium alone {lane 3} and lhcn solubilizcd with I~; NP40. hnmunoprec ip ihdcs with anli-gr~13() pcplidc antibody from Ihc NP40 lysatcs wcrc analyzed by S1)S PA(}[i and stll'~scqllcOl imnltlllObiolling with allli-phosphotyro SillC alllibod3.

phosphorylation of gpl3() following stimulation with II,-6-rclated cytokincs (lp ct al., 1992; Taga ctal . , 1992: Murakami ct al., 1993: Davis ct al., 1993; Yin ct al., 1993), we examined thc phosphorylation status of gp 13(I protein ill ES celts stimulated with D I A / L I F and the II ,-(~/slk-6R complex. NP4(I lysatcs from the stimu- latcd ccIls were subjected to immunoprecipitation with anli-mousc gp130 antibody, lmmunoprecipitates were analyzcd by SDS-PAGE and subsequcnt immunoblot- ling with anti-phosphotyrosinc antibody. As shown in Fig. 4B, the lk-(~/slL-6R eomplcx induced rapid tyro- sinc-phosphorylation of gpl30. In contrast, the gpl30- immunoprec ip i t a t e s p r e p a r e d from D I A / LIF- stimulated ES ceils contained tyrosinc-phosphorylatcd gpl30 alld a higher MW protcin which is likely to be I ) I A / I . I I : - R . Assoc iat ion of D I A / L I F - R with gpl30 and tyrosine phosphorylation of both of thesc molecules have been observed in other cell types on stimulation with I ) I A / L I F (Ip ct al., 1992; Taga et a[., 1992; Davis eI al., 1993). Our data strongly suggest that D I A / L I F - R in KS cells similarly associates with gpl30 in re- sponse to I ) I A / L I F and undergoes tyrosine phospho- rylation. However, l176/slL-6R-sl imulat ion of gpl30 ahmc, bypassing D I A / L I F - R activation, appears to be sufficient for the propagation of tile pluripotcntial phc- notype of t:~S cells.

3. Discuss ion

Fccder layers (/1 embryonic fibroblasts or condi- [ioncd media of particular cell lines are known to be required to support the maintenance of the pluripoten- till phenotype of ES cells (Evans and Kaufman, 1981;

Martin, 1981: Smith and Hoopcr, 1987). These cells produce several cytokincs including D I A / I ~ I I : , insulin-like grow, th factor-l I, translorming growth fac- tor-/3 and steel factor (Smith et al., 198bk Heath and Smith, 1988: Zscbo ctal . , 19g0). It has hccn shown |ha! among thcsc various factors only purified I ) I A / I J F can reversibly inhibit the diffcrcntiation of t:~S cells culturcd in vitro without feeders and sustain the nor- real dcvclopmental polentia[ of the cells. This suggcsts that D I A / L I F is a necessary and sufficient factor for the maintenancc of pluripotcntialily of KS cells (Wil- liams el al., 1988: Smith et al., 198;8: Smith, 1992). 111 this paper we have shown that other cytokines which can activate gp 13(I-associated signalling pathways in KS cells can also support ES ccll sclf-rcnewal and sustain pluripotcncy. Thus both CNTF and OSM act on L:,"; cells to maintain the undiffcrentiatcd phcnotype in a manner indistinguishable from that of I ) I A / L I F . Not all factors whosc rcccptors includc gpl30 are effective on ES cells, however. Intcrlcukin-l l did not suppress ES diffcrentiation in our assays (data not shown), pos- sibly indicating that KS cells lack an ll ,-l l-spcci[ic r c c c p t o r c o n l p o n e n [ .

I ) I A / L I F has bccn proposed to play an important and specific role in the self-renewal of stem cells in ea r lymousc embryos(Smith and Rathjen 19t)l: Smith ct al., 19t)2). l-towcvcr, rcccnt rcporls on l ) I A / l , l k dcficicnt mice gcncrated by gcnc targeting casI doubt on lhc function of D I A / L I F in viw~, sincc homozygous null embryos arc capable of normal development (Stewart et al., lq~.~2: Escary' et al., 1(~()3). The data presented here suggest that the cytokines ( 'NTF' or OSM or the combination of Ik-fl and sll,-6R could duplicate the activity of I ) I A / I J F " and compcnsatc for abscncc of thc latter in null mutants. Although the actions of CNTF arc generally considered to bc con- fined to neuronal cells, lhc effcclivc inhibition of ES cell differentiation at picomoh, r concentrations implies Ihal KS cells cxprcss the spccific ('NTF-l,t, tr subunit and arc therefore rcsponsivc to physiological levels of CNTF. Murinc OSM has not yct been charactcriscd but has been postulated to sharc ovcrlapping biological activity with D I A / L I F (Rose and Bruce, 1991 ). The production of I L-O by blastoeyst stagc naottsc cmhryos has been documcntcd (Murray c[ al., 1990)and the physioh)gical significance of the scflublc form of l l.-flR is indicated by its detection ill hu|nan and nlouse scrn (Honda c ta l . , 1t)92: Suzuki ct al., It)L)3). sllAd,t, prc- sent il1 scra is biologically active, i.e. it can inducc, when supplemented with rccombinant 11,-6, DNA syn- thesis in gpl30 cI)NA-transfectcd mouse pro-B cclls (Suzuki ct al., 1t~93: Narazaki et al., It)t)3). The concen- tration of serum slL-6R ill mice increases in accor- dance with age and it varies from strain to strain and between sexes. Half-maxinlal effect of the combination of recombinant slL-6R and 11.-6 on tile maintenance of

K- Yoshida et al. / Mechanisms of Det'elopment 45 (1994) 163-17l 169

pluripotentiality of ES cells in vitro was observed at concentrations of 250 n g / m l and 20 ng /ml , respec- tively. This seems likely to be within the physiological range, particularly if account is taken of the likely lower affinity of the cross-species interaction between human 1L-6/sIL-6R complex and murine gpl30. Two mechanisms have been reported for the production of sIL-6R: (1) format ion of alternatively spliced mRNA lacking the transmembrane domain and (2) proteolytic shedding from membrane-anchored IL-6R (Lust et al., 1992; Miillberg et al., 1993). It remains to be deter- mined whether early mouse embryos can produce slL- 6R or whether maternal sIL-6R can be transferred into developing embryos.

The signalling processes of both IL-6 and D I A / L l F in differentiated cell types have previously been shown to involve gpl30 and activation of tyrosine kinases (Ip et al., 1992; Taga et al., 1992; Davis et al., 1993; Murakami et al., 1993). It is worthy of comment that the membrane proximal signalling events which direct stem cell renewal in pluripotential ES cells appear indistinguishable from those which initiate differentia- tion in myeloid cells or mediate induction of acute phase protein synthesis in hepatocytes, for example. It is also noteworthy that the combination of IL-6 and sIL-6R or the complex of IL-6 and membrane-anchored IL-6R induces homodimerization of gpl30, whereas D I A / L I F stimulation results in the formation of het- e rodimers comprising gpl30 and D I A / L I F - R (Murakami et al., 1993; Davis et al., 1993). It has also been shown that IL-6 stimulation does not induce DIA/L1F-R-associat ion with gpl30 nor does D I A / LIF stimulation induce gpl30 homodimerization. Thus, the structure of the homodimer of gpl30 formed in response to IL-6 stimulation is distinct from that of the g p l 3 0 - D I A / L I F - R heterodimer formed on D I A / L I F binding. Our results in the present work suggest that formation of either gp 130-gp 130 homodimers or gp130- D I A / L 1 F - R heterodimcrs, although their structures differ, can initiate signalling processes in ES cells which result in the maintenance of their pluripotentiality. Thus although there is evidence that the D I A / L I F - R subunit can activate distinct signalling pathways from those activated by gpl30 (Tanigawa et al., 1993), this does not appear to be required for ES cell self-re- newal. It is now important to identify and characterize downstream molecules, such as gpl30-associated ki- nase(s), that mediate the signalling processes initiated by each of these two types of dimers.

4. Experimental procedures

4.1. Cells and recombinant factors

Several lines of ES cells, CGR2, CGRS, CP1, D3 and El4, were used in this study. Unless indicated,

they were maintained as previously described (Smith, 1991) on gelatin-coated plastic dishes or plates in DMEM or GMEM supplemented with 0.1 mM /3- mercaptoethanol, 10% FCS and 100 uni t s /ml routine D1A/LIF . The recombinant human factors used in these experiments are as previously detailed (Taga et al., 1992) except D I A / L I F which was purchased from Amrad and their concentrations were 10 n g / m l (oncostatin M), 1000 uni t s /ml ( D I A / L I F and IL-6) and 2.5 p ,g /ml (sIL-6R), unless specifically indicated. Recombinant routine D I A / L I F was prepared as de- scribed (Smith et al., 1988; Smith, 1991) and rat CNTF was obtained from Genzyme.

4.2. Detection of stem cell markers

ES cells cultured in gelatin-coated 24 well-plates were subjected to alkaline phosphatase staining using Sigma Diagnostic Kit No. 86 (Sigma) according to the manufacturer 's procedures. For flowcytometric analysis of SSEA-1 expression, ES cells seeded in 3.5 cm diam- eter dishes at a density of 4,500 cel ls /ml (2 ml /d ish) were cultured for 6 days under various conditions. Cells were harvested by using 0.125% trypsin, 0.01% EDTA in PBS and stained with anti-SSEA-1 mono- clonal antibody and fluorescein-conjugated anti-mouse lgM antibody. FACScan (Becton Dickinson) was used for analysis. For fluorescent and light microscopic anal- yses, ES cells were cultured in gelatin-coated Chamber Slides (Nunc) at an initial density of 5 × 104 cel ls /ml (1 ml / chamber ) for 7 days. Cells were fixed with methanol and analyzed directly by phase contrast mi- croscopy or by fluorescent microscopy after subsequent staining with the anti-SSEA-I antibody and fluores- cein-conjugated anti-mouse IgM antibody.

4.3. Production of ES cell chimaeras

Microinjection of ES cells into C57BL/6 host blas- tocysts was performed essentially as described by Bradley (1987). Injected blastocysts were allowed to re-expand in culture before transfer to the uteri of pseduopregnant MF1 recipients. Chimaeras were iden- tified among the resulting offspring by the presence of sandy coat color pigmentation.

4.4. RNA analysis

RNA preparation and RNase protection assays were performed as described (Rathjen et al., 1990a; Robert- son et al., 1993). For TISl l inductions, CP1 ES cells were plated onto gelatin-coated dishes at a density such that the cultures were semi-confluent the follow- ing day. Cells were incubated in GMEM plus 10% FCS without D I A / L I F for 1 h after washing with the same medium. Cells were then washed and incubated in

17t) Ix'. }e(,%'hld(l ('I (l/. / /il('['/l(llli.lJt#].l~ Q~ 1)~' '[+q)mc#11 45 ( / 0 9 4 ) ]¢~.? ] 7]

GMEM alone fl)r 6 -7 h. Medium was then replaced with G M E M either ahme or supplemented with D I A / H F (1,0(10 un i t s /ml ) or IL-6 (500 n g / m l ) and slL-6R (5 # g / m l ) . Forty min later, RNA was prepared by a modified method of Chomczynski and Sacchi (1987) and subjected to electrophoresis on a fo rmaldehyde / agarosc gel prior to transfer to membrane and hy- bridization at 65°(-? for 18 h (Church and Gi/bcrt, 1984). The probc was a 984 bp Xho l fragment encompassing the full coding region of T ISI I (Varnum et 21., 1991).

4.5. DN4 sy#lt/lesLs assay

5. Acknowledgements

We thank Davor Soltcr 1~,+1 + anti-SSEA-I, Harvey Herschman for the TISI I probc, Morag Robcrtson, Annette Dfiwel and Kcnji Yoshida for technical contri- butions, Kyoko Kubota and Keiko Ono for sccrctarial assistance and Frank Jol lnstol l and (J,-ahanl [3rown fo]

photographic reproductions. This work was supported by a Grant-in-Aid for Scicncc Rcscarch from the Min- istry of Education, Science and Culture. ,lapan, tile Human Frontier Scicncc Program and Uchara Mcmo rial Foundation.

ES cells were seeded in 96 well-platcs at a density of 2 × 104 ce l l s /ml (0.1 ni l /well) and cultured for 2 days tinder various conditions. Cells were then pulse-labeled with [3H]thymidine (0.5 taCt /wel l ) for 17 h and har- vested on glass filters. Incorporated radioactivity was measured by Beta Plate System (Pharmacia),

4. 6. hnmu#loprecipitatio#l

ES cells maintained in 9 cm diameter dishcs at a subconfluent density were cultured with methionine- free DMEM for 30 rain and metabolically labeled with [~S]mcthionine (0.2 mCi) in 2 ml each of mcthioninc- frcc DMEM for 1.5 h with occasional tilting. Labeled cells werc stimulated with either IL-6 plus slk-6R, sll+-6R alone, or medium alone (2 ml of DMEM, lOC;~ FCS) for 10 min at 37°(?. Cells were harvested with (-Tell Lifter (Costar Corp.) and NP40 lysates from the cells were immunoprecipitated with anti-human IL-6R monochmal antibody, MTI8 and analyzed by SI)S- PAGE and auloradiography as previously described (t t irata ct al., 198t): Taga et al., 1992).

4. 7. hmnt+##obh)tli#lg

ES cells which had been maintained in the presencc of D I A / L I F were washed and cultured in the absence of D I A / L I F for 7 h and stimulated with either D I A / LIF (1000 U / m l ) or 1L-6 (5 # g / m l ) plus slL-6R (2.5 # g / m l ) at 37°C for It) rain. Ceils were solubilized with 1% NP40 and the lysates subjected to immunoprccipi- tat ion with polyclonal anti-gpl30 peptide antibody, lm- munoprecipitatcs were analyzed by SDS-PAGE and subsequent immunoblotting with anti-phosphotyrosine antibody as described (Taga et al., 1992). Data were obtaincd by using Enhanced Chemi-Luminescent Dc- tection Kit (ECL System, Amersham) according to manulacturer's proccdures. Anti-gpl30 antiserum was obtained by immunizing a rabbit with a peptide, KKHIWPNVPDPSKSHIA, as described (Hirata ct al., 198;9). +rhe antiserum was affinity-purified on peptide- coupled Sepharose 413 prior to use.

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