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First published online June 13, 2005
Stem Cells Vol. 23 No. 8 September 2005, pp. 1192 -1199
doi:10.1634/stemcells.2004-0286; www.StemCells.com
© 2005 AlphaMed Press

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Homologous Feeder Cells Support Undifferentiated Growth and Pluripotency in Monkey Embryonic Stem Cells

Tianqing Lia,b,c, Shufen Wanga,b,c, Yunhua Xiea,b,f, Yongqing Lua,b,c, Xiuzhen Zhanga,b,c, Liu Wange, Shihua Yanga,b, Don Wolfd, Qi Zhoue, Weizhi Jia,b,f

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{at}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{at}ioz.ac.cn


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the present study, five homologous feeder cell lines were developed for the culture and maintenance of rhesus monkey embryonic stem cells (rESCs). Monkey ear skin fibroblasts (MESFs), monkey oviductal fibroblasts (MOFs), monkey follicular granulosa fibroblast-like (MFG) cells, monkey follicular granulosa epithelium-like (MFGE) cells, and clonally derived fibroblasts from MESF (CMESFs) were established and compared with the ability of mouse embryonic fibroblasts (MEFs) to support rESC growth. MESF, MOF, MFG, and CMESF cells, but not MFGE cells, were as good as or better than MEFs in supporting undifferentiated growth while maintaining the differentiation potential of the rESCs. In an effort to understand the unique properties of supportive feeder cells, expression levels for a number of candidate genes were examined. MOF, MESF, and MEF cells highly expressed leukemia inhibitory factor, ciliary neurotrophic factor, basic fibroblast growth factor, stem cell factor, transforming growth factor ß1, bone morphogenetic protein 4, and WNT3A, whereas WNT2, WNT4, and WNT5A were downregulated, compared with MFGE cells. Additionally, all monkey feeder cell lines expressed Dkk1 and LRP6, antagonists of the WNT signaling pathway, but not WNT1, WNT8B, or Dkk2. rESCs grown on homologous feeders maintained normal karyotypes, displayed the characteristics of ESCs, including morphology, alkaline phosphatase, Oct4, the cell surface markers stage-specific embryonic antigen (SSEA)-3, SSEA-4, tumor-related antigen (TRA)-1-60, and TRA-1-81, and formed cystic embryoid bodies in vitro that included differentiated cells representing the three major germ layers. These results indicate that the four homologous feeder cell lines can be used to support the undifferentiated growth and maintenance of pluripotency in rESCs.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
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) [1–6]. Self-renewal, the key characteristic of ESCs, entails suppression of differentiation during proliferation [7]. This fate choice is highly regulated by intrinsic signals and the extrinsic microenvironment [8]. Whereas it can be demonstrated that the use of feeder cells inhibits the spontaneous differentiation of primate ESCs in vitro [1–5], the identity of the essential self-renewal signals is currently unknown [9].

To date, prolonged propagation of rhesus monkey ESCs (rESCs) is achieved only by coculture with primary mouse embryonic fibroblasts (MEFs) serving as feeder cells [1]. However, there are disadvantages in using MEFs secondary to their limited proliferating abilities, interbatch variability [10, 11], and the possible introduction of mouse viruses and/or foreign proteins [8]. 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 [11–13], 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 [MESFs]), 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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Culture Media
Fibroblast culture medium (FCM) was Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Invitrogen Corporation, Carlsbad, CA, http://www.invitrogen.com) supplemented with 10% newborn bovine serum (HyClone, Logan, UT, http://www.hyclone.com) containing 1x penicillin-streptomycin.

Granulosa cell culture medium (GCM) contained basic medium (DMEM, 1 mM glutamine [Sigma, St. Louis, http://www.sigmaaldrich.com], 0.1 mM mercaptoethanol [Sigma], and 1x penicillin-streptomycin) and 10% fetal bovine serum (Gibco)

Culture medium for clonally derived cells (CCM) contained 10% MESFs (P3) 24 hour–conditioned culture medium (FCM) and 90% fresh medium. The fresh medium was composed of 85% basic medium and 15% fetal bovine serum (Gibco).

R366.4 rESCs (gift from Dr. James Thomson) were cultured in ECM composed of 90% basic medium and 10% HyClone defined fetal bovine serum (dFBS).

The rESC differentiation medium (DCM) contained 85% DMEM/F12 (1:1), 15% dFBS, 1 mM glutamine, 0.1 mM mercaptoethanol, and 1x nonessential amino acids.

Establishment of MESF, MOF, MFG, MFGE, and CMESF Feeder Layers
MESF and MOF feeder layers were aseptically established from the ear skin of a neonatal (1-week-old) and the oviduct of a juvenile (2-year-old) rhesus monkey, respectively. Skin and oviduct explants (1–2 mm3) were placed in 75-cm2 tissue culture flasks (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) containing FCM and incubated at 37°C in a 5% CO2 in air atmosphere. Five to eight days later, confluent primary monolayers were established.

Granulosa cells were obtained from the cumulus of immature oocytes recovered after ovarian stimulation and cultured in vitro to the mature MII stage. Individual cells were harvested after cumulus digestion with 1 mg/ml hyaluronidase. Cells were suspended in GCM, plated into flasks at a density of 1 x 105 cells per cm2, and cultured in the CO2 incubator. When the cultures neared or reached confluence (approximately 1 week), two distinct morphological cells (fibroblast-like and epithelium-like) appeared, and confluent cells were harvested after digestion with 0.125% trypsin. Trypsin action was considered complete when fibroblast-like cells rounded up. The cells were agitated manually with pipette in GCM and replated on new gelatin-coated plates. Epithelium-like cells that remained on the plates were expanded to confluency and harvested as described above. After four or five repetitions of this protocol, the purity of two cell types exceeded 95%. By this method, MFG and MFGE feeder layers were successfully established.

To obtain higher purity cell lines, single MESF (P3) cells were cloned. A single cell was plated into individual microdrops (100 µl) and cultured in CCM on gelatin-coated plastic, covered with mineral oil. After 3–4 weeks of culture, 8.57% (6/70) drops of inoculated cells contained a colony of cells. Then, confluent cells were digested with 0.125% trypsin and 0.02% EDTA, and passaged. After 2–3 months, six cloned cell lines (CMESF) were established.

rESC Culture
rESCs, initially cultured on MEFs [1], were plated onto homologous cell monolayers (passage 8 [P8] and mitotically inactivated by irradiation). Once rESC colonies grew to maximal size without the onset of visible differentiation, cells were digested with 10 mg/ml dispase (Gibco) and seeded onto newly prepared feeder cells.

Estimation of rESC Growth on Rhesus Monkey Feeders and MEFs
R366.4 ESCs grown on monkey feeders for 12 passages were digested by 1 mg/ml dispase for 3 minutes, and trypsin (0.25% in 0.04% EDTA) to single cells [14]. Five x 103 cells per cm2 were inoculated onto new feeders. After single rESCs were grown on the monkey feeders and MEFs for 12 days, ESCs were fixed in situ with 4% paraformaldehyde, and stained with Hoechst 33342 and stage-specific embryonic antigen (SSEA)-4 to allow quantification of colony and cell number. Colony formation rate was determined as the percentage of colony number divided by the total number of inoculated cells. Cell expansion fold in passages was counted as the number of cell number per colony multiplied by the number of undifferentiated colonies. Colony response to alkaline phosphatase (Apase) staining was scored as that typifying pure undifferentiated stem cells, mixed populations, or fully differentiated cells; the latter two were classified as differentiated colonies [15]. The cell differentiation rate was determined as the percentage of differentiated colonies divided by the total number of colonies.

Apase Staining and Karyotypic Analyses of rESCs
Cocultures used for Apase staining were established in either four- or six-well plates. Prior to analysis, adherent cell layers were fixed by the addition of 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 minutes. After a washing with PBS solution, Apase staining was performed using a kit containing BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/Nitro Blue Tetrazolium) as substrate. Before and after continuous culture for 15–20 passages (>3 months) on the monkey feeders, karyotyping analyses of rESCs were carried out as previously described [16]. To avoid disturbance of monkey feeders and their inclusion in the karyotype analysis, rESCs were digested and plated onto MEF feeders for two passages before analysis as reported before [12].

Immunohistochemical Staining
Cultured colonies, attached embryoid body (EB), or differentiated cells were fixed in situ with 4% paraformaldehyde in PBS for 20 minutes at room temperature. After blocking with 10% goat serum, the cells were stained with one of the following primary antibodies: SSEA-3, SSEA-4, SSEA-1, tumor-related antigen (TRA)-1-60, TRA-1-81, Oct-4, and nestin (all from Chemicon International, Temecula CA, http://www.chemicon.com); alpha fetoprotein (AFP) and albumin (polyclonal antibody; Sigma); and microtubule-associated protein 2 (MAP2), glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP) (polyclonal antibody; all from Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com). The cells were then rinsed three times with PBS and incubated for 30 minutes with fluorescein isothiocyanate (FITC)– or phycoerythrin (PE)–conjugated second antibody (Santa Cruz Biotechnology). Negative controls for each fluorophore-conjugated secondary antibody were carried out without the addition of the primary antibody, and no nonspecific binding of secondary antibodies was detected. The immunolabeled cells were examined using a confocal laser scanning system (LSM 510 META; Carl Zeiss, Jena, Germany, http://www.zeiss.com).

RNA Preparation and Gene Expression Analyses
Total RNA was extracted using a TRIZOL RNA isolation kit (Invitrogen Corporation). Potential contamination from genomic DNA was eliminated by DNase digestion. Cytoplasmic RNA (5 µg) from MEFs and MOF, MESF, and MFGE cells was reverse-transcribed to single-stranded cDNA. Aliquots of cDNA were used as a template for polymerase chain reaction (PCR) amplification with specific primer sets derived from the conserved sequences of the human, mouse, and/or rat genes [17, 18]. The sense and antisense primer sequences, corresponding cDNA PCR condition, and product sizes are shown in Table 1Go. Five µl of PCR products were separated on a 1.5% agarose gel and visualized by ethidium bromide staining. The relative expression levels of the detected genes from these cells were estimated visually by comparing relative band intensities with the expression level of housekeeping gene, GAPDH.


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  		Table 1. Polymerase chain reaction primers and conditions for gene analysis
 
ESC Differentiation In Vitro
rESCs were digested with 1 mg/ml dispase, suspended in ECM, and plated in six-well plates containing feeder cells that had been attached for 12 hours. After this incubation, rESCs remaining in the medium were collected by centrifugation, resuspended in DCM, and cultured in hanging drops for EB production (30 µl/ drop, 60 cells/µl). Three days later, aggregated simple EBs were transferred to bacteria culture plates to maintain continuous suspension cultures. Another 5–7 days later, cystic EBs were plated into six-well gelatin-coated plates and continuously cultured for 20 days. Differentiated rESCs were examined by immunocytochemistry or by morphology observations.

Statistical Analysis
The results are presented as means ± SEM. Statistical analysis was performed using the least significant difference test. Statistical significance was defined as p < .05.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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. 1AGo). 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. 1BGo–2FGo), and tested positive for the expression of Apase (Fig. 2AGo), SSEA-3 (Fig. 2BGo), SSEA-4 (Fig. 2CGo), TRA-1-60 (Fig. 2DGo), TRA-1-81 (Fig. 2EGo), and Oct-4 (Fig. 2FGo), but not for SSEA-1 (Fig. 2GGo). These cells also displayed normal karyotypes (42, XY), similar to that for the MEF control.



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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.

 


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  		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 2Go) on the basis of 800 colonies examined in four replicates. To evaluate the variation between batches of MEFs [14] 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.


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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
 
Gene Expression Analysis
The results are shown in Figure 3Go. 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 ß1 (TGFß1), 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.



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  		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. 4AGo) and then cystic EB formation after suspension culture for another 7 days (Fig. 4BGo). 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. 4CGo). After continuous culture for 10 days, AFP (Fig. 4GGo) and nestin (Fig. 4IGo) were detected in differentiated cells from EB. After 20 days, vascular-like structures (Fig. 4DGo), neuron-like cells (Fig. 4EGo), and muscle-like cells (Fig. 4FGo) were observed. Furthermore, albumin (hepatocyte marker, Fig. 4HGo), MAP2 (neuron marker, Fig. 4JGo), MBP (oligodendrocyte marker, Fig. 4KGo), and GFAP (astrocyte marker, Fig. 4LGo) 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.



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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.

 
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 [19, 20]. 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Large and efficient expansion is a precondition for the further study and application of rESCs. To date, establishment and culture of all rESC lines required a layer, so-called feeder cells from MEFs that supply components necessary to sustain the self-renewal of rESCs. However, the disadvantages in using MEFs are due to their limited proliferating abilities, interbatch variability [10, 11], and the possible introductions of mouse viruses and/or foreign proteins [8]. In the present study, we reported a new culture system to expand rESCs with rhesus monkey feeder cells. Our results showed that the rESCs could be undifferentiated growth on the four monkeys feeders (MESF, MOF, MFG, and CMESF), even on the high-passage feeders. The monkey ESCs maintained all the characteristics of rESCs, such as cell morphology, normal chromosomal karyotypes, typical surface markers expression, and the formation of cystic EBs in vitro that included differentiated cells representing the three major germ layers as previously reported in undifferentiated primate ESCs [1–5]. These results indicate that four homologous feeder cell lines can be used to support the undifferentiated growth of rESCs and maintain their pluripotency as well as human feeders can [11–13]. The use of homologous feeders could set the stage for the derivation and maintenance of ESCs without exposure to foreign proteins or viruses [11–13]. Our next step for future research will be to use monkey serum, instead of FBS and newborn calf serum, or even a serum substitute in culturing rESCs and establishing monkey feeders to completely avoid the contamination of foreign proteins.

Current culture conditions are suboptimal, and a significant percentage of primate ESCs died at each passage on MEFs, even when ESCs were passaged in a clump [14]. Furthermore, spontaneous differentiation occurred readily, and the culture system did not easily support clonal growth of primate ESCs, which led us to believe that autocrine growth factors produced by the primate ESCs themselves may be required for self-renewal of the ESC population [5]. However, in the study, the analysis of colony formation rate of single rESCs showed there was significant difference about colony formation rate on different monkey feeders and MEFs. Further comparative studies showed MOF was the preferable homologous feeder for rESC growth, followed by MESF, CMESF, and MFG (Table 2Go). Therefore, we supposed the growth of primate ESCs may be also related with microenvironment afforded by feeders.

Previous studies demonstrated that several signaling pathways, such as LIF, FGF, WNT, and TGF-ß pathways, play key roles in maintaining growth and self-renewal of human and mouse ESCs [21–24]. LIF, bFGF, SCF, TGF-ß1, BMP4, and WNT3A are able to support the continued proliferation, suppress the differentiation, or sustain self-renewal of ESCs and adult stem cells [25–33]. The analysis of WNT pathway genes shows that human ESCs (hESCs) express most WNT pathway genes and do not express any kind of the WNT ligands [21], which suggested that WNT ligands are secreted only by feeder to maintain the undifferentiated growth of hESCs. In the present study, we found LIF, bFGF, SCF, TGF-ß1, BMP4, and WNT3A were highly expressed in MOF and MESF cells and MEFs, but downexpressed in MFGE cells (Fig. 3Go). Those results might partly explain why MOF and MESF cells supported the undifferentiated growth of rESCs, whereas MFGE cells did not. Additionally, the higher level of WNT3A in MOF cells than that in other feeders may partly explain that MOF is the preferable homologous feeder for rESC growth.

WNT5A, WNT2, and WNT4 were highly expressed in MFGE cells, but downregulated in MOF and MESF cells and MEFs. It had been reported that these genes were overexpressed in human differentiating EBs and differentiated cells [21]. From these results it is supposed that WNT5A, WNT2, and WNT4 may not be essential for primate ESC renewal or may facilitate the differentiation of the ESCs. The lower level of WNT5A, WNT2, and WNT4 expressed in MOF cells also explained why the rESCs could grow better on the MOF feeder. It has been demonstrated that the WNT antagonist Dkk1 was required for adult bone marrow stem cells to re-enter the cell cycle [34]. LRP is a coreceptor for WNT, and at the same time, it is a specific, high-affinity receptor for Dkk1 and Dkk2 [35, 36]. Brandenberger et al. [21] reported that WNT1, WNT8B, Dkk1, and Dkk2 were not expressed in hESCs, but LRP6 equally expressed in hESCs and differentiated EBs. Our results also confirmed that all of the feeder cell lines expressed Dkk1 and LRP6, but not WNT1, WNT8B, and Dkk2. We proposed that WNT1, WNT8B, and Dkk2 might not be necessary for the growth of primate ESCs.

In summary, the four homogenous rhesus feeders were produced to replace MEFs supporting the undifferentiated growth of rESCs and maintain their pluripotency. The variation in the feeders’ abilities for rESC growth may be related with differences of some genes’ expression. However, these genes whose products supported the growth of primate ESCs were currently not identified. Separating the soluble and cell-associated components required for prolonged growth and expansion of primate ESCs was impossible in culture with mixed cell populations such as poorly characterized MEFs [11]. High-purified monkey feeders that maintain long-term and abundant expansion and have stable abilities to support the undifferentiated growth of rESCs will enable us to facilitate the research on primate ESCs.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received October 20, 2004; accepted for publication April 14, 2005.



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