标题: Homologous Feeder Cells Support Undifferentiated Growth and Pluripotency in Monk [打印本页] 作者: 江边孤钓 时间: 2009-3-5 10:49 标题: Homologous Feeder Cells Support Undifferentiated Growth and Pluripotency in Monk
a Kunming Primate Research Center, Chinese Academy of Sciences, Kunming, Yunnan, China; / f* s7 h9 N5 e) e) N: V$ t7 A3 I5 R/ i: `2 K/ P
b Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China;' G( \8 L* S: [+ U% V! ]: z% ]5 f
9 s5 Y3 z9 H6 L$ T8 c& n. g9 }c Graduate School, The Chinese Academy of Sciences, Beijing, China;) l, o8 Q% n& f2 i1 F U
% }1 v6 V" I: c6 [8 v% d1 e, V1 v; vd Oregon National Primate Research Center, Portland, Oregon, USA; " Q9 U5 v6 E+ O7 o 6 B$ n. r" [" L* ke Institute of Zoology, Chinese Academy of Sciences, Beijing, China; 5 c, |1 g% G# W& j3 m' a, @7 y) G p* A) L6 C, F3 K# c: }, X
f Yunnan Key Laboratory for Animal Reproductive Biology, Kunming, Yunnan, China " I" B, r$ Y2 i' e/ k' ~" G v5 ? ' w8 Q0 I4 |8 T7 T2 u. O/ ~( M( fKey Words. Embryonic stem cells ? Rhesus monkey feeders ? Stem cell markers ? Self-renewal ? Wnt signaling d, G0 W* W' S! X0 n
1 B. h- [4 d8 i) PCorrespondence: Weizhi Ji, Ph.D., Kunming Primate Research Center and Kunming Institute of Zoology, Chinese Academy of Sciences, 32 Jiaochang Donglu, Kunming, Yunnan, 650223, China. Telephone: 86-871-5139413; Fax: 86-871-5139413; e-mail: wji@mail.kiz.ac.cn; and Qi Zhou, Ph.D., Institute of Zoology, Chinese Academy of Sciences, Beijing 100086, China. Telephone: 86-10-62650042; e-mail: qzhou@ioz.ac.cn+ t8 v ~% \; q7 B' n
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ABSTRACT 1 M m: e3 q4 y. [9 |0 M0 Y. ~5 E) }$ |( e& Y& ^2 c7 d) v; i
Previous reports have indicated that both the derivation and maintenance of primate embryonic stem cells (ESCs) require the use of supporting cells, either as a feeder layer or as a source of conditioned medium and extracellular matrix (ECM) . Self-renewal, the key characteristic of ESCs, entails suppression of differentiation during proliferation . This fate choice is highly regulated by intrinsic signals and the extrinsic microenvironment . Whereas it can be demonstrated that the use of feeder cells inhibits the spontaneous differentiation of primate ESCs in vitro , the identity of the essential self-renewal signals is currently unknown . % B0 [) f, H6 {# C- n/ a5 Y' i4 K' s3 ~! c$ b5 m8 Q
To date, prolonged propagation of rhesus monkey ESCs (rESCs) is achieved only by coculture with primary mouse embryonic fibroblasts (MEFs) serving as feeder cells . However, there are disadvantages in using MEFs secondary to their limited proliferating abilities, interbatch variability , and the possible introduction of mouse viruses and/or foreign proteins . These shortcomings suggest that MEFs may not be the most appropriate feeder cells for this application. Furthermore, human ESC culture on human cells has been described recently , implying that homogenous cells can serve as feeder cells and prolong the undifferentiated growth of rESCs.6 b5 j& z' |+ n; o( M
, u6 Q) c, i# J$ ~4 m& f) mIn the present study, we developed five rhesus monkey feeder cell lines: the ear skin fibroblasts from a neonatal, 1-week-old monkey (monkey ear skin fibroblasts ), oviductal fibroblasts from ajuvenile (2-year-old) animal (MOFs), adult follicular granulosa fibroblast-like (MFG) cells, adult follicular granulosa epithelium-like (MFGE) cells, and clonally derived fibroblasts from the MESF (CMESFs). These cells were tested for their ability to support the culture and propagation of rESCs, compared with MEFs, and examined for the expression of candidate genes related to ESC growth, maintenance, and self-renewal. " f) H- c# b- k/ c2 M5 |2 J. e- c9 K; c' d ; t' E1 |$ \5 S3 e4 A3 n6 ^MATERIALS AND METHODS% ]) F1 g- F8 ^* T* c2 b
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Prolonged Expansion of rESCs Cultured on Rhesus Monkey Feeders1 f7 |9 W O% S! |# q
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Three MESF, two MOF, two MFG, two MFGE, and six CMESF cell lines were established as feeders. MESF, MOF, MFG, and CMESF cell lines were passaged every 2 days in the ratio of 1:3, whereas the MFGE cell line was done every 3–4 days in the ratio of 1:2. The feeders were irradiated and cryopreserved in liquid nitrogen with 10% dimethyl sulfoxide. Greater than 90% of the feeder cells survived after freezing and thawing as judged by Trypan Blue staining. rESCs growing for 15–20 passages on MESF, MOF, CMESF, and MFG cell lines, even at high passage numbers (CMESF: P20; MESF and MOF: P18; MFG: P12), remained completely undifferentiated. This allowed passaging every 7 days similar to that normally required for cells on MEFs. In contrast, rESCs did not survive when cultured on the MFGE cell line (Fig. 1A). Morphologically, the rESC colonies grown on monkey feeder cells had a large surface area with distinct boundaries, giving the colonies a more compact shape than that observed on MEFs. Under high magnification, individual rESCs grown on monkey feeders were small and round, with prominent nucleoli typical of rESCs on MEFs (Figs. 1B–2F), and tested positive for the expression of Apase (Fig. 2A), SSEA-3 (Fig. 2B), SSEA-4 (Fig. 2C), TRA-1-60 (Fig. 2D), TRA-1-81 (Fig. 2E), and Oct-4 (Fig. 2F), but not for SSEA-1 (Fig. 2G). These cells also displayed normal karyotypes (42, XY), similar to that for the MEF control.$ z9 g9 a6 z# k7 ~! e% t) z
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Figure 1. Morphology of rESCs grown on four rhesus monkey feeders and MEFs for 15 to 20 passages. (A) MFG feeder cells, which did not support rESC growth; (B) rESCs on MOF feeder cells; (C) rESCs on MFG feeder cells; (D) rESCs on CMESF feeder cells; (E) rESCs on MESF feeder cells; (F) rESCs on MEFs. Bars = 100 μm. Abbreviations: CMESF, clonally derived fibroblasts from MESF; MEF, mouse embryonic fibroblast; MESF, monkey ear skin fibroblast; MFG, monkey follicular granulosa fibroblast-like; MOF, monkey oviductal fibroblast; rESC, rhesus monkey embryonic stem cell.8 I0 Y- O$ i. m. b0 b4 R
9 U6 v1 N- D" A* A$ L, r+ MFigure 2. Characterization of undifferentiated rESCs grown on monkey feeders for 15 to 20 passages. MOF was used in these representative micrographs; however, similar results were obtained for rESCs grown on MFG, MESF, and CMESF feeder cells. Alkaline phosphatase activity (A), immunostaining for SSEA-3 (B) and SSEA-4 (C), TRA-1-60 (D) and TRA-1-81 (E), Oct-4 (F), and SSEA-1 (G). Bars = 50 μm (A–E), 100 μm (F, G). Abbreviations: CMESF, clonally derived fibroblasts from MESF; MESF, monkey ear skin fibroblast; MFG, monkey follicular granulosa fibroblast-like; MOF, monkey oviductal fibroblast; rESC, rhesus monkey embryonic stem cell; SSEA, stage-specific embryonic antigen; TRA, tumor-related antigen. 4 t% [5 V: `8 L9 o2 y . ~. U0 D3 `$ k- w- s& I5 M) ^rESC Growth , H8 `; P9 v, G7 G7 V Z( k' m$ t v( Q) o( z8 m2 _
After 12 days of single ESC culture, the colony formation rate, cell number per colony, cell expansion fold in passage, and differentiated rate were quantitated (Table 2) on the basis of 800 colonies examined in four replicates. To evaluate the variation between batches of MEFs and different cell lines of each type, three MEFs, three MESF, two MOF, two MFG, and three CMESF cell lines were examined, and similar results were achieved in all cases (data not shown). These results indicated that MESF, MOF, MFG, and CMESF cell lines were as good as or better than MEFs in supporting the undifferentiated growth of rESCs.: |$ F) l2 g+ A& I# t) v: j
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Table 2. Rhesus monkey feeders were compared with the abilities of MEFs to maintain the growth and self-renewal of rESCs / T8 I% O" }( k5 g, G2 h ' i9 n% U5 O, aGene Expression Analysis8 d( t" D3 J$ k/ p
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The results are shown in Figure 3. MOF, MESF, and MEF cells highly expressed leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF), stem cell factor (SCF), transforming growth factor ?1 (TGF?1), bone morphogenetic protein 4 (BMP4), and WNT3A, whereas WNT2, WNT4, and WNT5A were downregulated, compared with MFGE cells. Additionally, all feeder cell lines expressed Dkk1 and LRP6, antagonists of the WNT signaling pathway, but not WNT1, WNT8B, and Dkk2.$ L; } \3 H& {4 S
) m2 g) ]/ t; S8 L/ o8 KFigure 3. Representative analyses of gene expression in MEFs, MOF, MESF, and MFGE cells. Cytoplasmic RNA from MEFs and MOF, MESF, and MFGE cells was used to construct cDNA pools, and the expression of genes was examined by polymerase chain reaction. The lane number at the top of each figure indicates: MEFs (lane 1) and MOF (lane 2), MESF (lane 3), and MFGE (lane 4) cells. Abbreviations: MEF, mouse embryonic fibroblast; MESF, monkey ear skin fibroblast; MFGE, monkey follicular granulosa epithelium-like; MOF, monkey oviductal fibroblast.9 }, x2 x0 q8 v+ a
( p6 J4 Q& X! K' e$ gDifferentiation Potential ' ^& ^/ i; r; D: |/ y9 Y. z- B/ |+ i5 J4 _' |: n5 _# Z8 ?
rESCs grown on MOF cells for 15 passages were representative of the spontaneous differentiation that occurs when cells are removed from coculture with first EB formation (Fig. 4A) and then cystic EB formation after suspension culture for another 7 days (Fig. 4B). When placed on six-well gelatin-coated plates, EB adhered to the substrate and became flat cell masses that quickly proliferated (within 2 days) and outgrew (Fig. 4C). After continuous culture for 10 days, AFP (Fig. 4G) and nestin (Fig. 4I) were detected in differentiated cells from EB. After 20 days, vascular-like structures (Fig. 4D), neuron-like cells (Fig. 4E), and muscle-like cells (Fig. 4F) were observed. Furthermore, albumin (hepatocyte marker, Fig. 4H), MAP2 (neuron marker, Fig. 4J), MBP (oligodendrocyte marker, Fig. 4K), and GFAP (astrocyte marker, Fig. 4L) were observed in these differentiated cells. rESCs cultured on MESF, MFG, and CMESF cells also formed cystic EBs that included differentiated cells representing the three major germ layers.+ m ^7 Y7 L# } W! y# b2 n4 M
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Figure 4. In vitro differentiated rESCs grown on monkey feeder for 15 to 20 passages. rESCs grown on MOF feeder cells were used in these representative micrographs; however, similar results were obtained for rESCs grown on MFG, MESF, and CMESF feeder cells. Simple representative EB in hanging drop culture for 4 days (A); a cystic EB (B). (C): Within 2 days, EB adhered to the substrate and became flat cell masses that quickly proliferated and outgrew. Vascular-like structures (D); neuron-like cells (E); muscle-like cells (F); immunostaining of in vitro differentiation cells from rESCs grown on MOF for 20 passages (G–L); AFP in day 10 differentiated cells (G); albumin as hepatocyte marker in day 20 differentiated cells (H); nestin in day 10 differentiated cells (I); MAP2 as a neuronal-specific protein (J); MBP as an oligodendrocyte marker (K); GFAP as an astocyte marker (L). Blue Hoechst 33342 labeled nucleolus. Bars = 100 μm (A–I), 50 μm (J–L). Abbreviations: AFP, alpha fetoprotein; CMESF, clonally derived fibroblasts from monkey ear skin fibroblast; EB, embryoidbody; GFAP, glial fibrillary acidic protein; MBP, myelin basic protein; MESF, monkey ear skin fibroblast; MFG, monkey follicular granulosa fibroblast-like; MOF, monkey oviductal fibroblast; rESC, rhesus monkey embryonic stem cell. ! X& J! g. U7 u$ |2 i; j9 p" J2 N O( Y5 ]+ ]
To compare the pluripotency of rESCs cultured on different feeders, 10 ng/ml bFGF was added to the serum-free medium to induce day-9 EB differentiation into neural progenitors (NPs) as described previously . The differentiation rate was determined after Hoechst 33342 and nestin staining of 10-day cultures based on 4,500 to 5,000 cells examined in three replicates. There were no significant differences in differentiation rates of rESCs grown on MESF, MOF, MFG, or CMESF cells, or MEFs at 62 ± 5.3%, 64 ± 8.1%, 60.2 ± 7.2%, 58 ± 6.1%, and 59 ± 9.2%, respectively (p .05). 2 f/ ?5 G. q, h/ l8 O0 u4 [' v5 Q5 ?. C3 ^- g
DISCUSSION ! d- Y& J5 p% u; B 6 d3 | T# c( N9 R5 MThis work was supported by research grants from Major State Research Development Program 2004CCA01300, G200016108 and 2001cb510100, The Chinese Academy of Sciences KSCX1-05, Chinese National Science Foundation 30370166, and Yunnan Nature Science Foundation 2001C0009Z. Tianqing Li and Shufen Wang contributed equally to this study.; w0 A3 w7 b* @# H/ P" y
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