Homologous Feeder Cells Support Undifferentiated Growth and Pluripotency in Monkey Embryonic Stem Cells
http://www.100md.com
《干细胞学杂志》
a Kunming Primate Research Center, Chinese Academy of Sciences, Kunming, Yunnan, China;
b Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China;
c Graduate School, The Chinese Academy of Sciences, Beijing, China;
d Oregon National Primate Research Center, Portland, Oregon, USA;
e Institute of Zoology, Chinese Academy of Sciences, Beijing, China;
f Yunnan Key Laboratory for Animal Reproductive Biology, Kunming, Yunnan, China
Key Words. Embryonic stem cells ? Rhesus monkey feeders ? Stem cell markers ? Self-renewal ? Wnt signaling
Correspondence: 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
ABSTRACT
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 .
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.
In 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.
MATERIALS AND METHODS
Prolonged Expansion of rESCs Cultured on Rhesus Monkey Feeders
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.
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.
Figure 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.
rESC Growth
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.
Table 2. Rhesus monkey feeders were compared with the abilities of MEFs to maintain the growth and self-renewal of rESCs
Gene Expression Analysis
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.
Figure 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.
Differentiation Potential
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.
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.
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).
DISCUSSION
This 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.
REFERENCES
Thomson JA, Kalishman J, Golos TG et al. Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci U S A 1995;92:7844–7848.
Thomson JA, Kalishman J, Golos TG et al. Pluripotent cell lines derived from common marmoset (Callithrix jacchus) blastocysts. Biol Reprod 1996;55:254–259.
Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147.
Suemori H, Tada T, Torii R et al. Establishment of embryonic stem cell lines from cynomolgus monkey blastocysts produced by IVF or ICSI. Dev Dyn 2001;222:273–279.
Reubinoff BE, Pera MF, Fong CY et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000;18:399–404.
Xu C, Inokuma MS, Denham J et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001;19:971–974.
Burdon T, Smith A, Savatier P. Signaling, cell cycle and pluripotency in embryonic stem cells. Trends Cell Biol 2002;12:432–438.
Odorico JS, Kaufman DS, Thomson JA. Multilineage differentiation from human embryonic stem cell lines. STEM CELLS 2001;19:193–204.
Czyz J, Wiese C, Rolletschek A et al. Potential of embryonic and adult stem cells in vitro. Biol Chem 2003;384:1391–1409.
Park JP, Kim SJ, Oh EJ et al. Establishment and maintenance of human embryonic stem cells on STO, a permanently growing cell line. Biol Reprod 2003;69:2007–2014.
Richards M, Fong CY, Chan WK et al. Human feeders support prolonged undifferentiated growth of human inner cell mass and embryonic stem cells. Nat Biotechnol 2002;20:933–936.
Cheng L, Hammond H, Ye ZH et al. Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture. STEM CELLS 2003;21:131–142.
Hovattal O, Mikkolal M, Gertow K et al. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod 2003;18:1404–1409.
Amit M, Carpenter MK, Inokuma MS et al. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev Biol 2000;227:271–278.
Chambers I, Colby D, Robertson M et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 2003;113:643–655.
Freshney RI. Culture of Animal Cells: A Manual of Basic Techniques, 4th Ed. New York: Wiley-Liss, Inc., 2000; 235–245.
Kayo T, Aillison DB, Weindruch R et al. Influences of aging and caloric restriction on the transcriptional profile of skeletal muscle from rhesus monkey. Proc Natl Acad Sci U S A 2001;98:5093–5098.
Lu SJ, Quan C, Li F et al. Hematopoietic progenitor cells derived from embryonic stem cells: analysis of gene expression. STEM CELLS 2002;20:428–437.
Zhang SC, Wernig M, Duncan ID et al. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol 2001;19:1129–1133.
Kuo HC, Pau KYF, Yeoman RH et al. Differentiation of monkey embryonic stem cells into neural lineages. Biol Reprod 2003;68:1727–1735.
Brandenberger R, Wei H, Zhang S et al. Transcriptome characterization elucidates signaling networks that control human ES cell growth and differentiation. Nat Biotechnol 2004;22:707–716.
Amit M, Shariki C, Margulets V et al. Feeder layer- and serum-free culture of human embryonic stem cells. Biol Reprod 2004;70:837–845.
Ying QL, Nichols J, Chambers I et al. BMP induction of ID proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 2003;115:281–292.
Qi XX, Li TG, Hao J et al. BMP4 supports self-renewal of embryonic stem cells by inhibiting mitogen-activated protein kinase pathways. Proc Natl Acad Sci U S A 2004;101:6027–6032.
Matsuda T, Nakamura T, Nakao K et al. STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells. EMBO J 1999;18:4261–4269.
Niwa H, Burdon T, Chambers I etal. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev 1998;12:2048–2060.
Matsui Y, Zesbo K, Hogan BL. Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 1992;70:841–847.
Taipale J, Beachy PA. The Hedgehog and Wnt signaling pathways in cancer. Nature 2001;411:349–354.
Reya T, Duncan AW, Ailles L et al. A role for Wnt signaling in self-renewal of haematopoietic stem cells. Nature 2003;423:409–414.
Willert K, Brown JD, Danenberg E et al. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 2003;423:448–452.
Aubert J, Dunstan H, Chambers I et al. Functional genes screening in embryonic stem cells implicates Wnt antagonism in neural differentiation. Nat Biotechnol 2002;20:1240–1245.
Ding S, Wu TYU, Brinker A et al. Synthetic small molecules that control stem cell fate. Proc Natl Acad Sci U S A 2003;100:7632–7637.
Sato N, Meijer L, Skaltsounis L etal. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 2004;10:55–63.
Gregory CA, Singh H, Perry AS et al. The Wnt signaling inhibitor dick-kopf-1 is required for reentry into the cell cycle of human adult stem cells from bone marrow. J Biol Chem 2003;278:28067–28078.
Mao BY, Wu W, Li Y et al. LDL-receptor-related protein6 is a receptor for Dickkopf proteins. Nature 2001;411:321–325.
Wehrli M, Dougan ST, Caldwell K et al. Arrow encodes an LDL-receptor-related protein essential for Wingless signalling. Nature 2000;407:527–530.
Soloviev MM, Ciruela F, Chan WY et al. Mouse brain and muscle tissues constitutively express high levels of homer proteins. Eur J Biochem 2000;267:634–639.(Tianqing Lia,b,c, Shufen )
b Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China;
c Graduate School, The Chinese Academy of Sciences, Beijing, China;
d Oregon National Primate Research Center, Portland, Oregon, USA;
e Institute of Zoology, Chinese Academy of Sciences, Beijing, China;
f Yunnan Key Laboratory for Animal Reproductive Biology, Kunming, Yunnan, China
Key Words. Embryonic stem cells ? Rhesus monkey feeders ? Stem cell markers ? Self-renewal ? Wnt signaling
Correspondence: 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
ABSTRACT
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 .
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.
In 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.
MATERIALS AND METHODS
Prolonged Expansion of rESCs Cultured on Rhesus Monkey Feeders
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.
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.
Figure 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.
rESC Growth
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.
Table 2. Rhesus monkey feeders were compared with the abilities of MEFs to maintain the growth and self-renewal of rESCs
Gene Expression Analysis
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.
Figure 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.
Differentiation Potential
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.
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.
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).
DISCUSSION
This 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.
REFERENCES
Thomson JA, Kalishman J, Golos TG et al. Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci U S A 1995;92:7844–7848.
Thomson JA, Kalishman J, Golos TG et al. Pluripotent cell lines derived from common marmoset (Callithrix jacchus) blastocysts. Biol Reprod 1996;55:254–259.
Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147.
Suemori H, Tada T, Torii R et al. Establishment of embryonic stem cell lines from cynomolgus monkey blastocysts produced by IVF or ICSI. Dev Dyn 2001;222:273–279.
Reubinoff BE, Pera MF, Fong CY et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000;18:399–404.
Xu C, Inokuma MS, Denham J et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001;19:971–974.
Burdon T, Smith A, Savatier P. Signaling, cell cycle and pluripotency in embryonic stem cells. Trends Cell Biol 2002;12:432–438.
Odorico JS, Kaufman DS, Thomson JA. Multilineage differentiation from human embryonic stem cell lines. STEM CELLS 2001;19:193–204.
Czyz J, Wiese C, Rolletschek A et al. Potential of embryonic and adult stem cells in vitro. Biol Chem 2003;384:1391–1409.
Park JP, Kim SJ, Oh EJ et al. Establishment and maintenance of human embryonic stem cells on STO, a permanently growing cell line. Biol Reprod 2003;69:2007–2014.
Richards M, Fong CY, Chan WK et al. Human feeders support prolonged undifferentiated growth of human inner cell mass and embryonic stem cells. Nat Biotechnol 2002;20:933–936.
Cheng L, Hammond H, Ye ZH et al. Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture. STEM CELLS 2003;21:131–142.
Hovattal O, Mikkolal M, Gertow K et al. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod 2003;18:1404–1409.
Amit M, Carpenter MK, Inokuma MS et al. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev Biol 2000;227:271–278.
Chambers I, Colby D, Robertson M et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 2003;113:643–655.
Freshney RI. Culture of Animal Cells: A Manual of Basic Techniques, 4th Ed. New York: Wiley-Liss, Inc., 2000; 235–245.
Kayo T, Aillison DB, Weindruch R et al. Influences of aging and caloric restriction on the transcriptional profile of skeletal muscle from rhesus monkey. Proc Natl Acad Sci U S A 2001;98:5093–5098.
Lu SJ, Quan C, Li F et al. Hematopoietic progenitor cells derived from embryonic stem cells: analysis of gene expression. STEM CELLS 2002;20:428–437.
Zhang SC, Wernig M, Duncan ID et al. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol 2001;19:1129–1133.
Kuo HC, Pau KYF, Yeoman RH et al. Differentiation of monkey embryonic stem cells into neural lineages. Biol Reprod 2003;68:1727–1735.
Brandenberger R, Wei H, Zhang S et al. Transcriptome characterization elucidates signaling networks that control human ES cell growth and differentiation. Nat Biotechnol 2004;22:707–716.
Amit M, Shariki C, Margulets V et al. Feeder layer- and serum-free culture of human embryonic stem cells. Biol Reprod 2004;70:837–845.
Ying QL, Nichols J, Chambers I et al. BMP induction of ID proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 2003;115:281–292.
Qi XX, Li TG, Hao J et al. BMP4 supports self-renewal of embryonic stem cells by inhibiting mitogen-activated protein kinase pathways. Proc Natl Acad Sci U S A 2004;101:6027–6032.
Matsuda T, Nakamura T, Nakao K et al. STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells. EMBO J 1999;18:4261–4269.
Niwa H, Burdon T, Chambers I etal. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev 1998;12:2048–2060.
Matsui Y, Zesbo K, Hogan BL. Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 1992;70:841–847.
Taipale J, Beachy PA. The Hedgehog and Wnt signaling pathways in cancer. Nature 2001;411:349–354.
Reya T, Duncan AW, Ailles L et al. A role for Wnt signaling in self-renewal of haematopoietic stem cells. Nature 2003;423:409–414.
Willert K, Brown JD, Danenberg E et al. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 2003;423:448–452.
Aubert J, Dunstan H, Chambers I et al. Functional genes screening in embryonic stem cells implicates Wnt antagonism in neural differentiation. Nat Biotechnol 2002;20:1240–1245.
Ding S, Wu TYU, Brinker A et al. Synthetic small molecules that control stem cell fate. Proc Natl Acad Sci U S A 2003;100:7632–7637.
Sato N, Meijer L, Skaltsounis L etal. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 2004;10:55–63.
Gregory CA, Singh H, Perry AS et al. The Wnt signaling inhibitor dick-kopf-1 is required for reentry into the cell cycle of human adult stem cells from bone marrow. J Biol Chem 2003;278:28067–28078.
Mao BY, Wu W, Li Y et al. LDL-receptor-related protein6 is a receptor for Dickkopf proteins. Nature 2001;411:321–325.
Wehrli M, Dougan ST, Caldwell K et al. Arrow encodes an LDL-receptor-related protein essential for Wingless signalling. Nature 2000;407:527–530.
Soloviev MM, Ciruela F, Chan WY et al. Mouse brain and muscle tissues constitutively express high levels of homer proteins. Eur J Biochem 2000;267:634–639.(Tianqing Lia,b,c, Shufen )