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RAPID COMMUNICATION |
a Tokyo Metropolitan Institute of Technology, Systems Engineering Science, Hino, Tokyo, Japan;
b Okayama University Graduate School of Medicine and Dentistry, Department of Pathology, Okayama, Okayama, Japan;
c Hino Municipal Hospital, Hino, Tokyo, Japan;
d Kibi International University, Health Science, Takahashi, Okayama, Japan;
e Kanagawa Academy of Science and Technology, Kawasaki, Kanagawa, Japan
Key Words. Primate ES cells • Feeder layer • Human placenta • Teratoma
Correspondence: Kanji Miyamoto, Ph.D., Department of Systems Engineering Science, Tokyo Metropolitan Institute of Technology, Asahigaoka 6-6, Hino, Tokyo 191-0065, Japan. Telephone: 81-42-585-8641; Fax: 81-42-585-8641; e-mail: kmiyamot{at}cc.tmit.ac.jp
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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We evaluated the growth of primate ES cells (cynomolgus monkey ES cells; CMK6) on human amniotic epithelial (HAE) feeder cells and human chorionic plate (HCP) feeder cells derived from human placentas. Primate ES cells were used because they were of less ethical concern and human ES cells could not be used without the permission of the Japanese Government. The primate ES cells have many biological characteristics similar to human ES cells [8,9]. As the cynomolgus and Rhesus monkeys are closely related to humans, and because they are widely used for medical research, cynomolgus monkey ES cells would be valuable for preclinical research before the clinical usage of human ES cells [7].
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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MEF Feeder
Primary cells were cultured from 14-day C57BL/6 mouse strain embryos. Feeder cells were prepared as needed by thawing the cells and treating them with mitomycin-C.
Preparation of HAE and HCP Feeder Layers, and Feeder Layers Treated with Diluted Ammonia Solution (DAS; 0.28%–0.09%)
Human placentas were obtained with the written, informed consent of pregnant women who were negative for HTLV-I, HIV-I, and hepatitis B. We were recognized for the appropriate use of human placenta by the ethical and safety committee of the Tokyo Metropolitan Institute of Technology. Human AE and CP membranes were surgically separated from the placenta, incubated in 0.25% trypsin-EDTA (ethylenediaminetetraacetic acid; GIBCO) at 37°C for 30 minutes, and then washed with phosphate buffer solution (PBS). Thereafter, the cells were digested with 0.05% collagenase type 1 (Sigma, St. Louis) at 37°C for 30 minutes and washed three times with PBS. The HAE or HCP cells were placed in 25-cm2 tissue culture flasks (BD Falcon, Bedford, MA) containing 90% (v/v) Dulbecco’s Modified Eagle Medium (D-MEM) and 1 xpenicillin-streptomycin mixture, 10% KSR or fetal calf serum (GIBCO), and incubated at 37°C in 5% CO2 in air for 3 days. The cultured monolayers of HAE and HCP cells were treated with mitomycin-C (Sigma) for 3 hours, then by 0.25% trypsin-EDTA (GIBCO), to produce single-cell suspensions of feeder cells that were cultured in 3-mL dishes for 3 days. The full-seated HAE and HCP cells were used for feeder layers. The confluent HAE and HCP cells were further treated with DAS at room temperature for about 10 minutes and then washed three times with PBS. The treated HAE and HCP cells were used for feeder layers.
CMK6 Growth on Feeder Layers
CMK6 ES cells dissociated by treatment of 0.25% trypsin-EDTA solution were plated onto confluent MEF, HAE, and HCP feeder cells, along with the feeder cells treated with DAS, and then cultured in a medium consisting of 80% (v/v) F-12/D-MEM, 20% KSR or 10% inactivated human cord serum, 0.1 mM 2-mercaptoethanol, 1 mM Na-pyruvate, 2 mM l-glutamine, 0.1 mM nonessential amino acids, and 1xpenicillin-streptomycin mixture.
Immunostaining of Feeder Cells
To identify epithelial cells or mesenchymal cells within the feeder cells, cultured HAE and HCP feeder cells were immunostained as follows. The cells were cultured in a medium consisting of 80% (v/v) F-12/DMEM, 20% KSR, 0.1 mM 2-mercaptoethanol, 1 mM Na-pyruvate, 2 mM l-glutamine, 0.1 mM nonessential amino acids, and 1xpenicillin-streptomycin mixture. The cells were fixed with 4% para-formaldehyde solution (Nacalai Tesque, Kyoto, Japan) at room temperature for 10 minutes and then washed with PBS. Subsequently, the fixed feeder cells were reacted with 1:50 diluted monoclonal mouse antihuman cytokeratin 18 antibodies (reaction with epithelial cells) or monoclonal mouse antivimentin clone V9 antibodies (reaction with mesenchymal cells) (DakoCytomation, Carpinteria, CA), and detected by immunocytochemistry, using either an avidin or biotin horseradish peroxidase complex (Vectastain ABC system; Vector Laboratories, Burlingame, CA). To determine doubling times of HEA and HCP feeder cells, 5 x104 trypsin-EDTA solution and collagenase type 1 digested cells were seeded into a 0.1% gelatin-coated 3.5-cm dish.
Immunostaining of CMK6 Cells on Feeder Cells
CMK6 ES cells cultured on confluent HAE feeder layers were fixed with 2% glutaraldehyde solution at room temperature for 30 minutes, washed three times with PBS and methyl alcohol-acetic acid solution (3:1 v/v) at room temperature for 10 minutes, washed three times with PBS, treated with 0.5% Triton X-100 (Katayama, Chemical, Osaka, Japan) solution at room temperature for 30 minutes, and finally washed three times with PBS. Alkaline phosphatase activity was detected using the Vector Red Alkaline Phosphatase Substrate Kit 1 (Vector Laboratories). SSEA-1 and SSEA-4 (Chemicon Int., Temecula, CA) were detected in primate ES cells on MEF feeder cells, HEA and HCP feeder cells by immunocytochemistry, using a specific primary monoclonal antibody and either an avidin or biotin horseradish peroxidase complex (Vectastain ABC system).
Teratoma Formation
After 18 passages on HAE feeder cells, 5 x105 CMK6 ES cells were inoculated into the hind leg of severe combined immunodeficient (SCID) mice, and teratomas were recovered 7 weeks later. Tumors were embedded in paraffin and histologically examined after hematoxylin and eosin staining. HAE feeder cells (1 x106) inoculated into the same sites of SCID mice as controls did not develop tumors.
Reverse Transcription Polymerase Chain Reaction (RT-PCR) of Oct-4
Total RNA was extracted from CMK6 ES cells using the RNeasy Micro Kit (Qiagen Int., Valencia, CA) and reverse transcription was performed using the One Step RT-PCR kit (Qiagen Int.). Cynomolgus monkey Oct-4 gene primers (sense, 5'-GGACACCTGGCTTCGGATT-3'; antisense, 5'-TTCGCTTTCTCTTTCGGGC-3'), human Oct-4 gene primers (sense, 5'-CGTGAAGCTGGAGAAGGA-GAAGTG-3'; antisense, 5'-AAGGGCCGCAGCTTACA-CATGTTC-3'), and ß-actin internal standard primers (sense, 5'-TGGCACCACACCTTCTAGAATGAGC-3'; antisense, 5'GCACAGC TTCTCCTTAATGTCACGC-3') were used, and the PCR cycles consisted of a reverse transcription reaction at 50°C for 30 minutes, initial PCR activation step at 94°C for 15 minutes, followed by 94°C for 30 seconds, 67°C for 45 seconds, and 68°C for 1.5 minutes for 35 cycles. Negative-control PCR reactions were carried out under the same conditions for monkey and human Oct-4 genes, except that the reverse transcription reaction was performed at 50°C for 30 minutes. Positive-control PCR reactions were carried out using primers for ß-actin. The PCR product was electrophoresed through a 2% TBE (Tris-borate-EDTA) agarose gel stained with 0.1 µg/mL ethidium bromide. Gels were visualized on a UV transilluminator.
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RESULTS AND DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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). HAE feeder cells have supported the growth of undifferentiated CMK6 ES cells for at least 18 passages. CMK6 ES cell colonies grown in this manner require passage about every 8 days and remain morphologically intact. CMK6 ES cell colonies on HAE feeder cells and HAE feeder cells treated with DAS appeared to be more homologous with undifferentiated clones. CMK6 ES cells on HAE feeder cells were also cultured with F-12/D-MEM containing 10% inactivated human cord serum instead of 20% KSR. The CMK6 ES cells showed the same morphological characteristics and growth behaviors.
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), stage-specific embryonic antigen (SSEA)-1 negative (Fig. 2H), SSEA-4 positive (Fig. 2I), the transcription factor Oct-4 expression positive by RT-PCR, and they displayed the normal karyotype, 40, XY. Oct-4 expression in CMK6 ES cells was assessed using Rhesus monkey Oct-4 primers [15] and human Oct-4 primers [13]. The RT-PCR product bands were 697 bp and 241 bp, respectively (Fig. 3). Rhesus monkey Oct-4 primers were demonstrated to work on a cynomolgus monkey by Mitalipov et al. [15], since this Oct-4 mRNA sequence has more than 98% homology with the human Oct-4 sequence. The positive control used for the RT-PCRs was CMK6 ES cells on MEF feeder cells (Fig. 2E, G, and Fig. 3).
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) and had the characteristics of epithelial cells. In contrast, HCP feeder cells have the characteristics of mesenchymal cells (Fig. 2D, C). The doubling time for HCP feeder cells was about 4.5 days, but HAE feeder cells were slower growing, dividing only 1.5 times within 2 weeks.
The cultured CMK6 ES cells on HAE feeder cells produced typical immature teratomas in vivo after injection into SCID mice. The teratoma had abundant, unambiguous derivatives of all three embryonic germ layers, including immature neural tissues with melanin, cartilage, muscle, and glands (Fig. 4).
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). But CMK6 ES cells cultured on cell-free feeder matrices such as gelatin, laminin, fibronectin, collagen type 1 coated dishes, and Matrigel were shown to differentiate in these culture media (Fig. 2J). These data suggest that human feeder layers are superior to the matrices [12,13].
We found that both HAE and HCP feeder cells in the presence of animal-free culture media could support primate ES cells in an undifferentiated state. The HAE and HCP feeder cells may secrete some proteins in culture medium, or they may maintain some membrane proteins for undifferentiated growth of ES cells. Koizumi et al. [17] reported eight growth factors (epidermal growth factor, keratinocyte growth factor, hepatocyte growth factor, basic fibroblast growth factor, transforming growth factor alpha [TGF-
], TGF-ß1, TGF-ß2, and TGF-ß3) and two growth receptors (keratinocyte growth factor receptor and hepatocyte growth factor receptor) in preserved human amniotic membrane. Recently, Sakuragawa et al. [18] characterized neuron-like cells that expressed choline acetyltransferase and acetylcholine among HAE cells. Tsai et al. [19] have reconstructed damaged corneas by transplantation with autologous limbal epithelial cells cultured on HAE feeder cells. Considering that HAE cells have no major histocompatibility complex (MHC) class II and fewer MHC class I molecules, no significant graft versus host reaction should occur in patients. Human placenta is quite novel and important because it would provide a relatively available source of feeders for the growth of human ES cells for therapeutic purposes that are also free of ethical complications. Considering the similarity of primate and human ES cells, the feeder cells should be effective on human ES cells. We are now planning to culture human ES cells on the HAE or HCP cells.
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REFERENCES |
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X174 DNA/Hinf I markers. Lane 1: CMK6 ES cells on HAE feeder layers by use of human Oct-4 primers; Lane 2, CMK6 ES cells on HAE feeder layers by use of monkey Oct-4 primers. Lane 3: CMK6 ES cells on HCP feeder layers by use of human Oct-4 primers. Lane 4: CMK6 ES cells on HCP feeder layers by use of monkey Oct-4 primers. Lane 5: CMK6 ES cells on MEF feeder layers by use of human Oct-4 primers. Lane 6: CMK6 ES cells on MEF feeder layers by use of monkey Oct-4 primers. Lane 7: ß-actin of CMK6 ES cells on HAE feeder layers. Lane 8: Negative control CMK6 ES cells on HAP feeder layers by use of human Oct-4 primers. Human Oct-4, 241-bp product; monkey Oct-4, 697-bp product; ß-actin, 400-bp product.