标题: Essential Roles of Sphingosine-1-Phosphate and Platelet-Derived Growth Factor in [打印本页] 作者: 江边孤钓 时间: 2009-3-5 10:47 标题: Essential Roles of Sphingosine-1-Phosphate and Platelet-Derived Growth Factor in
a Monash Institute of Medical Research, Laboratory of Embryonic Stem Cell Biology, Australian Stem Cell Centre, Monash University, Clayton, Australia; , B( C5 f+ m1 r, t. j# [# H' O1 C8 i: S
b Hanson Institute, Division of Human Immunology, Institute of Medical and Veterinary Science, Adelaide, Australia7 Q+ w6 t4 V* k* A6 D" f1 N* }
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Key Words. Human embryonic stem cells ? Sphingosine-1-phosphate ? Platelet-derived growth factor ? Lysophosphatidic acid6 B+ T0 o+ {# K( H
/ D* f8 s. E) X8 v/ ]- sCorrespondence: Alice P谷bay, Ph.D., Monash Institute of Medical Research, Laboratory of Embryonic Stem Cell Biology, Australian Stem Cell Centre, Building 75, STRIP Monash University, Wellington Road, Clayton VIC 3800, Australia. Telephone: 61-39-271-1143; Fax: 61-9271-1198; e-mail: alice.pebay@med.monash.edu.au. U( o% _3 P4 w7 R( E
3 Y7 a. Y+ }3 K/ ^5 T9 SABSTRACT- T# T a5 U1 {. O
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Human embryonic stem cells (hESCs) derived from human blastocysts have generally been cultivated on feeder layers of primary mouse embryonic fibroblasts (MEFs) in media supplemented with fetal calf serum (FCS). However, serum contains a wide variety of biologically active compounds that might adversely affect hESC growth and differentiation. Thus, cultivation of cells in FCS complicates experimental approaches to defining the intracellular mechanisms required for hESC maintenance. Furthermore, there is a potential biosafety concern if cells cultured in animal sera are subsequently used for implantation into humans. Alternative approaches to this culture system have been described, such as the use of a complex serum replacement, knockout serum replacement (KSR) plus basic fibroblast growth factor (bFGF) . However, KSR still contains an undefined mixture of animal proteins. Although mouse embryonic stem (ES) cells are maintained in the pluripotent state by leukemia inhibitory factor (LIF), hESCs do not respond to LIF . The aim of this study was to identify specific factors in serum responsible for its beneficial effect on the growth of hESCs. % W( i) H8 j7 G6 ~6 w" ?. U: v2 x* e, k, d. h
Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are bioactive lysophospholipids that are released by activated platelets and present in serum . These lysophospholipids act on a wide range of cells and regulate numerous cellular functions, including proliferation and differentiation, from the early stages of embryonic development . Most of their effects are mediated by specific G-protein–coupled receptors: S1P1/Edg-1, S1P2/Edg-5/Gpcr13/H218/AGR16, S1P3/Edg-3, S1P4/Edg-6, S1P5/Edg-8, LPA1/Edg-2/rec.1.3/vzg-1/Gpcr26/Mrec1.3, LPA2/Edg-4, and LPA3/Edg-7/ RP4-678I3/HOFNH30 . Moreover, S1P is also considered an intracellular second messenger with as-yet undefined intracellular targets . Despite the use of FCS to support mouse and human ES cell growth for many years, the potential role for lysophospholipid signaling in stem cell renewal has not been examined previously. Platelet-derived growth factor (PDGF) is another serum component widely described as a potent mitogen, also shown to prevent apoptosis . The PDGF family is comprised of dimeric isoforms of polypeptide chains A, B, C, and D that bind two tyrosine kinase receptors, PDGFR- and PDGFR-?. PDGF can activate sphingosine kinases (SPKs), leading to a transient increase in intracellular S1P concentration, held to be responsible for PDGF-induced cell proliferation or survival in different cell types .* g8 g$ A* N4 e! U+ ^* @% H
% f" q+ w1 ~' k2 n# u1 o5 v# }3 F: gMATERIALS AND METHODS$ m/ w- |! O( g9 |, ^5 n
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hESCs expressed mRNA transcripts for LPA receptors (Fig. 2A) and mRNA transcripts and the corresponding proteins for three S1P receptors, S1P1, S1P2, and S1P3 (Figs. 2B, 2C), whereas these cells did not express mRNA for S1P4 and S1P5 (data not shown). hESCs also expressed mRNA transcripts for PDGFR- and PDGFR-? as well as the corresponding proteins (Figs. 2D–2J). Consistent with previous reports , we found that MEF expressed S1P1, S1P2, S1P3, LPA1, LPA2, PDGFR-, and PDGFR-? (data not shown).. `' D% H" Q9 e
% i% p4 ~5 D$ q& u. e7 {' \( E$ ZFigure 2. hESCs are targets of S1P, LPA, and PDGF. Reverse transcription–polymerase chain reaction for lysophospholipid receptors (A, B), PDGFR- (alpha), and PDGFR-? (beta) (D) with ( ) or without (–) reverse transcriptase. (C): Western blot analysis of S1P receptors. Immunostaining of hESCs with (E, H) Hoechst 33342, (F) PDGFR-, or (I) PDGFR-? and (G, J) GCTM-2 antibodies. Scale bars = 50 μm. Abbreviations: hESC, human embryonic stem cell; LPA, lysophosphatidic acid; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; S1P, sphingosine-1-phosphate.) v" b7 p# i& ]; R0 ?9 Q2 a6 f
" w; w! v5 G& r/ q o4 l; T( zWhen hESCs were grown on MEFs in a serum-free culture medium, they spontaneously differentiated into a morphologically diverse array of cell types. After 2 weeks in a serum-free medium, LPA (up to 50 μM) had no obvious effect on size or morphology of hESC colonies, whereas in the presence of either S1P or PDGF-AB (PDGF), the colonies appeared flatter and less-differentiated compared with controls. However, the combination of S1P and PDGF seemed to induce a more striking inhibition of spontaneous differentiation in the hESCs. To quantify these effects, we developed an enzyme-linked immunosorbent assay (ELISA)–based assay to measure expression of the stem cell surface antigen GCMT-2 using the embryonal carcinoma cell line GCT27C4 (Fig. 1A). We then incubated cells for 2 weeks with different agonists. Cells treated with S1P or PDGF showed greater reactivity with GCTM-2 than controls (S1P, 141% ± 41% signal of controls; PDGF, 215% ± 42% signal of controls), whereas hESCs treated with both S1P PDGF showed 447% ± 124% (n = 3, p 5 ?9 B5 A6 e7 g6 [' f3 _
" O7 E/ k7 y5 s9 S1 VBecause SPK is a key molecule in PDGF signaling pathways and has been shown to be directly activated by ERK1/2 , we examined its involvement in hESC differentiation. Thus, we verified the presence of SPK transcripts in hESCs and showed expression of SPK-1 and SPK-2 mRNA (Fig. 1F). We next investigated whether PDGF modulates SPK activity in hESCs. PDGF enhanced in a time-dependent manner the SPK activity in hESCs (Fig. 1G). This effect lasted for at least 60 minutes, with SPK activity increasing 1.6-fold over basal levels (75.3 ± 3.9 pmol/min per mg, n = 3) after 30 minutes of incubation (Fig. 1G). In contrast, PDGF did not induce a statistically significant activation of SPK in MEF (data not shown). Moreover, a 12-day treatment of hESCs with the SPK inhibitor DMS (Figs. 1E, 1H) blocked the effect of S1P PDGF, suggesting an essential involvement of SPK in the maintenance of hESCs in an undifferentiated state. Because MEF-SPK was not activated by PDGF, these data also indicate that the effect of PDGF on hESCs is direct and not feeder cell–mediated. Moreover, when hESCs were grown on Matrigel for 7 days, in the presence or absence of S1P PDGF, the GCTM-2 expression level was higher in the presence of these two compounds compared with the one observed in the control condition, strongly suggesting a direct effect of S1P PDGF on hESCs (Fig. 1I).0 _5 c/ P6 E8 u2 I K- b
+ H0 |/ t" o+ [1 WUsing flow cytometry, we confirmed the ELISA results reported above and showed that the percentage of GCTM-2 cells in hESC colonies cultivated for 1 week in the presence of S1P PDGF was slightly lower than that of cells grown in the presence of KSR but much higher than in serum-free medium (65.3% ± 0.9% vs. 88.1% ± 3.6%, n = 4; control , 18.6% ± 6.4%, n = 3; Figs. 1J, 1K). The precise levels of GCTM-2 cells in KSR or S1P PDGF varied from experiment to experiment but were always at least two times higher than controls. hESCs cultivated for 1 week with S1P PDGF had similar cell-cycle phase distributions to those cultivated in KSR, as measured by BrdU incorporation (respectively, 46.5% ± 5.4% and 46.3% ± 3.1% of the GCTM-2 cells were in S phase after 2 hours of labeling with BrdU, n = 3, Figs. 1J, 1K), indicative of a primary effect on hESC maintenance rather than an effect on cell-cycle progression. 9 j, L4 P$ w$ M5 S; j6 U2 g8 U3 W# q9 W- z
Long-term cultivation and assessment of expression of stem cell markers confirmed successful maintenance of hESCs in a serum-free medium in the presence of S1P PDGF. The hESC cultures grown in S1P PDGF formed colonies that were smaller and thinner compared with controls grown in serum, and they were therefore more fragile when passaged by mechanical dissociation. HES-2, -3, and -4 cells have been grown in a serum-free medium supplemented with S1P PDGF for, respectively, 16, 83, and 11 passages or approximately 80, 415, and 55 population doublings (Fig. 3A). Reverse transcription–PCR studies showed that S1P/PDGF-treated hESCs expressed the mRNA for Oct-4 and cripto (Fig. 3B), and immunostaining showed reactivity with antibodies anti–GCTM-2, Oct-4, TG-30 (recognizing CD9), and Tra-1-60 (Figs. 3C–3F, supplemental online Fig. 1). All three hESC lines tested retained a normal karyotype (Fig. 3G, supplemental online Fig. 1) and formed teratomas containing tissues representative of the three embryonic germ layers after inoculation in the testis capsule of SCID mice (Figs. 3H, 3I). Moreover, S1P/PDGF-treated HES-3 cells responded to noggin treatment and neuronal induction with formation of neuronal cells, as ascertained by immunostaining for nestin, ?-tubulin, and NF-200 (Figs. 3J–3L). Altogether, these data demonstrate that hESCs grown in the presence of S1P PDGF retain the normal characteristics of hESCs propagated in serum. Even if the use of this defined medium does not eliminate the feeder cell requirement, these findings provide a basis for the development of a fully defined hESC culture system.+ k/ M* k: h2 j& m; l8 T" m
) m* o4 n8 p: PFigure 3. Characterization of human embryonic stem cells. (A): HES-3 cells grown in the presence of S1P PDGF (p14). (B): Reverse transcription–polymerase chain reaction using mRNA from HES-3 cells grown in the presence of S1P PDGF (p6) using specific primers for Oct-4, cripto, with ( ) or without (–) reverse transcriptase. Immunostaining of HES-3 cells grown in the presence of S1P PDGF (p13) with (C) GCTM-2, (D) Oct-4, (E) TG-30, or (F) TRA-1-60. (G): Karyotyping of HES-3 cells grown in the presence of S1P PDGF. Histology of teratoma with cartilage and squamous epithelium (H), glandular epithelium, and pigmented cells (I) after injection of HES-3 (p6) into severe combined immunodeficiency mice. Neuronal differentiation assessed by (J) nestin, (K) ?-tubulin, and (L) neurofilament 200. Scale bars = 50 μm. Abbreviations: PDGF, platelet-derived growth factor; S1P, sphingosine-1-phosphate. 5 j$ H2 p: e/ O# z7 h0 x 5 ^1 l ~7 |! G3 uCONCLUSION$ V8 m' m+ c* H, w' ]9 R
1 R( v" i2 K( } c IA.P. and R.C.B.W. contributed equally to this work. This study was supported by Monash University, ES Cell International, and the National Institutes of Health (NIGMS GM68417). We are grateful to Drs. S. Hawes and R. Mollard for helpful discussions and to Dr. P. Andrews for providing TRA-1-60. / C8 r7 {& a8 R+ K0 P/ r% B3 n/ l" R( @+ ~. y
DISCLOSURES) }4 i4 S4 i8 J) n1 G8 v
+ h( x6 Y2 o" a3 D- DM.F.P. owns stock in and within the past 2 years performed contract work for ES Cell International. 4 }/ a& x' i; G8 a H2 s! K8 O5 A% O6 z2 `) |! J
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