: 31 4 2004 Kor J Fertil Steril, Vol 31, No 4, 2004, 12 1 2, 1,2 2 2 1,2 1 1 1 1 1,2 Human Amniotic Fluid Cells Support Expansion Culture of Human Embryonic Stem Cells Hee Sun Kim 1,2, Hye Won Seol 2, Hee Jin Ahn 2, Sun Kyung Oh 1,2, Seung Yup Ku 1, Seok Hyun Kim 1, Young Min Choi 1, Jung Gu Kim 1, Shin Yong Moon 1,2 1 Department of Obstetrics and Gynecology, 2 Institute of Reproductive Medicine and Population, Medical Research Center, College of Medicine, Seoul National University, Seoul, Korea Objective: This study was performed to evaluate the possibility of prolonged culture of human embryonic stem cells (hesc; SNUhES2) on human amniotic fluid cells (hafc), which had been storaged after karyotyping Method: The hafc was prepared for feeder layer in the presence of Chang's medium and STO medium (90% DMEM, 10% FBS) at 37 in a 5% CO 2 in air atmosphere Prior to use as a feeder layer, hafc was mitotically inactivated by mitomycin C The hescs on hafc were passaged mechanically every seven days with ES culture medium (80% DMEM/F12, 20% SR, bfgf) Results: The hafc feeder layer support the growth of undifferentiated state of SNUhES2 for at least 59 passages thus far SNUhES2 colonies on hafc feeder appeared slightly angular and flatter shape as compared with circular and thicker colonies observed with STO feeder layer and showed higher level with complete undifferentiation in seven days Like hesc cultured on STO feeders, SNUhES2 grown on hafc expressed normal karyotype, positive for alkaline phosphatase activity, high telomerase activity, Oct-4, SSEA-3, SSEA-4, Tra-1-60 and Tra-1-81 and formed embryoid bodies (EBs) Conclusion: The hafc supports undifferentiated growth of hesc Therefore, these results may help to provide a clinically practicable method for expansion of hesc for cell therapies Key Words: Human embryonic stem cells (hesc), Human amniotic fluid cells (hafc), Feeder layer, Undifferentiation, Expansion of hesc (human embryonic stem cells, hesc) (inner cell mass, ICM), (karyotype),, :, ) 110-744 28, Tel: (02) 2072-2384, Fax: (02) 3672-7601, e-mail: shmoon@snuackr * 21 (SC11011) - 261 -
(cell therapy) (fetal abnormalities) 1 3 (prenatal genetic diagnosis) 11,12 (mouse embryonic fibroblast, MEF) octamer-binding transcription (feeder layer) factor-4 (Oct-4), 1,2 RNA (retrovirus) (coculture) 4 (mo- use embryonic stem cell, mesc), gelatin leukemia inhibitory factor (LIF) (IVF-ET) 5 10 LIF 6 Xu 7 extracellular matrix Matrigel (conditioned media), 10 ml (1000 rpm, 8 ) Chang's (Irvine scientific,, Richards 8 (fetal Santa Anna, CA, USA) 1 ml muscle) (fetal skin) 35 mm (adult fallopian tuba) (epithelial cell) 05 ml 37, 5% CO 2,, Amit 4 Hovatta 9 (foreskin) 2 ml, Cheng 10 (adult bone ma- rrow) 4 5 025% trypsin-edta (human amniotic fluid cells, hafc) (genetic alteration) - 262 - (SNUhES2), 13
, 70 80% bfgf 02, µm Ma- 150 trigel 35 mm mitomycin C (001 mg/ml), Chang's, 10% Fetal Bovine Serum (HyClone, Utah, USA), penicillin streptomycin Dulbecco's Modified Eagle Medium (DMEM, GIBCO), (cell surface marker) AP, SSEA-1, 3 4 Tra 1-60, 1-81 STO (ATCC, USA), Oct-4 telomerase PCR 71 Oh (SNUhES2) 14 alkaline phosphatase (AP), Oct-4, stage-specific embryonic antigen-3, 4 (SSEA-3, 4) 500 4, (46,XX) 5 ml (DMEM/F12, 20% SR, 1% non- 14 essential amino acid, 01 M β-mercaptoethanol, 05% penicilline/streptomycin) 01% gelatin mitomycin C mitomycin C, (colony) glass knife 100 200 7 neurofilament heavy chain (NFH) keratin DMEM/F12 (GIBCO) RT-PCR primer, 20% serum replacement (SR, GIBCO), sequences PCR Amylase (for- 04 ng/ml basic fibroblast growth factor (bfgf, Invi- ward: 5'-GCTGGGCTCAGTATTCCCCAAATAC-3', retrogen), 1% non-essential amnio acid, 01 mm β-merca- verse: 5'-GACGACAATCTCTG-3'), albumin (forward: ptoethanol, 05% penicilline/streptomycin 5'-CCTTTGGCACAATGAAGTGGGTAACC-3', reve- rse: 5'-GACGACAATCTCTGACCTGAGTAGC-3'), CMP (forward: 5'-ATGACTGTGAGCAGGTGTGCATCAG- Mitomycin C (2 10 6 cells) 25 3', reverse: 5'-CTGGTTGATGGTCTTGAAGTCAGCCcm 2 flask 3'), enolase (forward: 5'-TGACTTCAGTCGCCTGATG- FBS DMEM ATCCC-3', reverse: 5'-TGCGTCCAGCAAAGATTGC- - 263 - (three germ layer) (derivatives) (endoderm) amylase albumin, (mesoderm) cartilage matrix protein (CMP) enolase (ectoderm)
CTTGTC-3'), NFH (fowaard: 5'-TGAACACAGACGC- 12, TATGCGCTCAG-3', reverse: 5'-CACCTTTATGTGA- GTGGACACAGAG-3'), keratin (forward: 5'-AGGAA- (over- ATCATCTCAGGAGGAAGGGC-3', reverse: 5'ATCT- growth) mitomycin C CAGGAGGAAGGGC-3', reverse: 5'-AAAGCACAGA- mitomycin C TCTTCGGGAGCTACC-3') PCR 94 30, 68 30 primer mitomycin C 72 30 30 PCR 2% agarose gel, mitomycin C mitomycin C (prolife- 9, ration) A B C D Figure 1 Morphology of SNUhES2 cell line grown on haf feeder layer (A) SNUhES2 P71-5 cell colony at day 2 on hafc treated with mitomycin C (B) SNUhES2 P71-5 cell colony at day 7 on hafc treated with mitomycin C (C) SNUhES2 P71-5 cell colony at day 2 on hafc non-treated with mitomycin C (D) SNUhES2 P71-5 cell colony at day 7 on hafc non-treated with mitomycin C - 264 -
A B Figure 2 Results of karyotyping(a) SNUhES2 P71-25 on hafc treated with mitomycin C: 46,XX (B) SNUhES2 P71-25 on hafc non-treated with mitomycin C: 46,XX (Figure 2A) (Figure, 2B) STO (46,XX),, mitomycin C (Figure 1A, B) mitomycin C (Figure 1C, D), (Figure 5A, B) Mitomycin C (Figure 5C) - 265 - AP (Figure 3A, G) SSEA-4 (Figure 3F, L), Tra-1-60 (Figure 3B, H), Tra-1-81 (Figure 3C, I), SSEA-1 (Figure 3D, J) SSEA-3 (Figure 3E, K) telomerase (Figure 4A) Oct-4 (Figure 4B),
A B C D E F G H I J K L Figure 3 Immunocytochemical staining results of cell surface markers for detecting undifferentiation state of hesc grown on hafc; (A) (F) SNUhES2 P71-25 on hafc treated with mitomycin C (G) (L) SNUhES2 P71-25 on hafc non-treated with mitomycin C (A, G) AP (+) (B, H) Tra-1-60 (+) (C, I) Tra-1-81 (+) (D, J) SSEA-1 ( ) (E, K) SSEA-3 (+) (F, L) SSEA-4 (+) mitomycin C 4 5 Matrigel (Figure 6) - 266 -
A A B C B Figure 4 Detection of telomerase activity and Oct-4 (A) telomerase activity Lane 1: SNUhES2 cells grown on hafc treated with mitomycin C, Lane 2: Heat inactivated control of lane 1 sample, Lane 3: SNUhES2 cells on hafc non-treated with mitomycin C, Lane 4: Heat inactivated control of lane 3 sample, Lane 6: hafc, Lane 7: Heat inactivated control of lane 6, Lane 8: Positive control (B) Oct-4 expression of SNUhES2 grown on hafc Lane 1, 3, 5: beta- actin of lane 2, 4 and 6, respectively, Lane 2: SNUhES2 cells grown on hafc treated with mitomycin C, Lane 4: SNUhES2 cells on hafc nond ih i i C L 6 hafc Figure 5 Morphology and gene expression in embryoid bodies (A) EBs formed with SNUhES2 P71-5 cells at day 7 on hafc treated with mitomycin C (B) EBs formed with SNUhES2 P71-5 cells at day 7 on hafc non-treated with mitomycin C (C) RT-PCR results for detection of three germ layer markers: Endoderm (amylase and albumin), mesoderm (CMP and enolase) and ectoderm (keratin and NFH) Lane 1 is undifferentiated hesc grown on hafc treated with mitomycin C, lane 2 is EB for day 5, lane 3 is EB for day 10 and lane 4 is hafc as negative control - 267 - (fetal bovine serum, FBS)
Figure 6 Morphology of colony of SNUhES2 cell on day 7 grown on feeder free condition (magnification: 1,2 1998 Thomson 1 RNA 4 extracellular matrix,, (soluble factor), 59 Mitomycin C (primate) 16,17, mi- tomycin C Cheng 10 AP, SSEA-4, TRA-1-60, TRA-1-81, SSEA-3, SSEA-1 (suspension),,, mitomycin C mitomycin C mitom- - 268-30, 10 serum-free, bfgf serum replacement (SR) 18 SR 2000 Xu 7 laminin, collagen, fibronectin matrigel extracellular matrix mitomycin C, mitomycin C
ycin C Cheng SSEA-4 TRA-1-60 mitomycin C TRA-1-81 flow cytometer, early passage, late passage (gene expression) mitomycin C microarray mitomycin C, (cytotoxicity) (carcinogen), mito- matirx, SR, transforming growth factor β1 mycin C (TGFβ1), LIF bfgf mitomycin C Amit 4 2 SR 3 3 3 (embryonic) (fe- tal), 23,24 Hovatta 9,, Richards 19 11, 50, 90% (embryonic stem cell, ESC), 80% Carpenter 20 Rosler 21 Xu 7 Thomson 1-269 - Amit 22 fibronectin (epithelioid), (amniotic fluid cell) (fibroblast) 13 Richards 8 mesenchymal (transition) (embryonic germ cell, EGC) (embryonic carcinoma cell, ECC) Oct-4, 25,26 telom-
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