: Kor. J. Fertil. Steril., Vol. 31, No. 4, 2004, , 1, , ,2 Human Amniotic Fluid Cells Support Expansion Culture of Hum

Size: px

Start display at page:

Download ": Kor. J. Fertil. Steril., Vol. 31, No. 4, 2004, , 1, , ,2 Human Amniotic Fluid Cells Support Expansion Culture of Hum"

은주 동
5 years ago
Views:

1 : Kor J Fertil Steril, Vol 31, No 4, 2004, , 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),, :, ) , Tel: (02) , Fax: (02) , shmoon@snuackr * 21 (SC11011)

2 (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) % trypsin-edta (human amniotic fluid cells, hafc) (genetic alteration) (SNUhES2), 13

3 , 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 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 ( cells) 25 3', reverse: 5'-CTGGTTGATGGTCTTGAAGTCAGCCcm 2 flask 3'), enolase (forward: 5'-TGACTTCAGTCGCCTGATG- FBS DMEM ATCCC-3', reverse: 5'-TGCGTCCAGCAAAGATTGC (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-

growth) mitomycin C CAGGAGGAAGGGC-3', reverse: 5'-AAAGCACAGA- mitomycin C TCTTCGGGAGCTACC-3')

(prolife- 9, ration) A B C D Figure 1 Morphology of SNUhES2 cell line grown on haf feeder layer

colony at day 7 on hafc treated with mitomycin C (C) SNUhES2 P71-5 cell colony at day 2 on hafc

4 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, primer mitomycin C 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

(Figure 1C, D), (Figure 5A, B) Mitomycin C (Figure 5C) - 265 - AP (Figure 3A, G) SSEA-4 (Figure 3F, L), Tra-1-60

5 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) 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

6 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)

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,

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

7 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 (fetal bovine serum, FBS)

8 Figure 6 Morphology of colony of SNUhES2 cell on day 7 grown on feeder free condition (magnification: 1, 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 , 10 serum-free, bfgf serum replacement (SR) 18 SR 2000 Xu 7 laminin, collagen, fibronectin matrigel extracellular matrix mitomycin C, mitomycin C

9 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 (embryonic) (fe- tal), 23,24 Hovatta 9,, Richards 19 11, 50, 90% (embryonic stem cell, ESC), 80% Carpenter 20 Rosler 21 Xu 7 Thomson 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-

10 erase 27 AP, SSEA-4, TRA-1-60 TRA-1-81, telomerase 13 Oct-4 telo- merase (Figure 4A) (in vivo) teratoma,, extracellular matrix (factor) cytokine (growth factor) bfgf bone morphogenic protein 4 (BMP4) 1 Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz, MA, Swiergiel JJ, Marshall VS, et al Embryonic stem cell lines derived from human blastocysts Sci- ence 1998; 282: , 2 Reubinoff BE, Pera MF, Fong CY, Trounson A, Bo- 28 ngso A Embryonic stem cell lines from human bla- stocysts: Somatic differentiation in vitro Nat Biote- chnol 2000; 18: proteomics 3 Pera MF, Reubinoff B, Trounson A Human embr- yonic stem cells J Cell Sci 2000; 13: Amit M, Margulets V, Segev H, Shariki K, Laevsky I, Coleman R, et al Human feeder layers for human, embryonic stem cells Biol Reprod 2003; 68: Williams R, Hilton D, Pease S, Wilson T, Stewart C, Gearing D, et al Myeloid leukemia inhibitory factor maintains the developmental potential of embryonic stem cells Nature 1988; 336: Lim KWE, Bodnar A Proteome analysis of condi-, tioned medium from mouse embryonic fibroblast fe- eder layers which support the growth of human em- bryonic stem cell Proteomics 2002; 2: , proteomics proteomics (factor)

11 7 Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, et al Feeder-free growth of undifferentiated human embryonic stem cells Nat Biotechnol 2001; 19: Richards M, Fong CY, Chan WK, Wong PC, Bongso A Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells Nat Biotechnol 2002; 20: Hovatta O, Mikkola M, Gertow K, Stromberg AM, Inzunza J, Hreinsson J, et al A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells Hum Reprod 2003; 18: Cheng L, Hammond H, Ye Z, Zhan X, Dravid G Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture Stem Cells 2003; 21: Milunsky A Amniotic fluid cell culture In Milunsky, A (ed) Genetic Disorder and the fetus Plenum Press; New York, 1979 pp Hoehn H, Salk D Morphological and biochemical heterogeneity of amniotic fluid cells in culture Methods Cell Biol 1982; 26: Prusa AR, Marton E, Rosner M, Bernaschek G, Hengstschlager M Oct-4 expressing cells in human amniotic fluid: A new source for stem cell research? Hum Reprod 2003; 18: Oh SK, Kim HS, Ahn HJ, Seol HW, Kim YY, Park YB, et al Derivation and characterization of new human embryonic stem cell lines, SNUhES1, SN- UhES2 and SNUhES3 Stem Cells In press Chomczynski P, Sacchi N Single-step method of RNA isolation by acid guanidinium thiocyanatephenol-chloroform extraction Anal Biochem 1987; 162: Thomson JA, Kalishman J, Golos TG, Durning M, Harris CP, Becker RA, et al Isolation of a primate embryonic stem cell line Proc Natl Acad Sci USA 1995; 92: Thomson JA, Kalishman J, Golos TG, Durning M, Harris CP, Hearn JP Pluripotent cell lines derived from common marmoset (Callithrix kacchus) blastocysts Biol Reprod 1996; 55: Amit M, Carpenter MK, Inokuma MS, Chiu CP, Harris CP, Waknitz MA, et al Clonally derived human embryonics stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture Dev Biol 2000; 227: Richards M, Tan S, Fong CY, Biswas A, Chan WK, Bongso A Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells Stem Cells 2003; 21: Carpenter MK, Rosler ES, Fisk GJ, Brandenberger R, Ares X, Miura T, et al Properties of four human embryonic stem cell lines maintained in a feederfree culture system Dev Dyn 2004; 229: Rosler ES, Fisk GJ, Ares X, Irving J, Miura T, Rao MS, et al Long-term culture of human embryonic stem cells in feeder-free conditions Dev Dyn 2004; 229: Amit M, Shariki C, Margulets V, Itskovitz-Eldor J Feeder layer- and serum-free culture of human embryonic stem cells Biol Reprod 2004; 70: Gosden CM Amniotic fluid cell types and culture Br Med Bull 1983; 39: Prusa AR, Hengstschlager M Amniotic fluid cells and human stem cell research: A new connection Med Sci Moni 2002; 8: Donovan PJ High Oct-ane fuel powers the stem cell Nature Genet 2001; 29: Pesce M, Scholer HR Oct-4: Gatekeeper in the beginnigs of mammalian development Stem Cells 2001; 19: Mosquera A, Fernandez JL, Campos A, Goyanes VJ, Ramiro-Diaz JR, Gosalvez J Simultaneous decrease of telomere length and telomerase activity with ageing of human amniotic fluid cells J Med Genet 1999; 36: Schuldiner M, Yanuka O, Itskovitz-Elder J, Melton DA, Benvenisty N Proc Natl Acad Sci USA 2000; 97:

Microsoft Word - 2-최영민.DOC

Microsoft Word - 2-최영민.DOC 종 설 대한생식의학회지 : 제 34 권제 4 호 2007 서울대학교의학연구원인구의학연구소 1, 의과대학산부인과학교실 2 김윤영 1 오선경 1,2 최영민 1,2 * Feeder Cells for Culture of Human Embryonic Stem Cells Yoon Young Kim 1, Sun Kyung Oh 1,2, Young Min Choi 1,2