标题: Human-Serum Matrix Supports Undifferentiated Growth of Human Embryonic Stem Cell [打印本页] 作者: 江边孤钓 时间: 2009-3-5 10:49 标题: Human-Serum Matrix Supports Undifferentiated Growth of Human Embryonic Stem Cell
a Centre for Stem Cell Biology and Developmental Genetics, Institute of Human Genetics, University of Newcastle, Newcastle upon Tyne, United Kingdom;3 f |7 S' M$ h8 R4 t
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b School of Biological and Biomedical Sciences, University of Durham, Durham, United Kingdom;& L# E8 o0 ], |( l$ V; N/ v5 q
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c Institute of Human Genetics, University of Newcastle, Newcastle upon Tyne, United Kingdom 6 D% G8 b4 @( _- R+ Q2 y2 {6 }% H( T1 L8 W4 f( j0 Y
Key Words. Human embryonic stem cells ? Pluripotency ? Differentiation ? Feeder-free, b& K" ]1 o# B% M* V1 z
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Correspondence: M. Stojkovic, Ph.D., Centre for Stem Cell Biology and Developmental Genetics, Institute of Human Genetics, University of Newcastle, Newcastle upon Tyne, U.K. Telephone: 44-191-241-8638; Fax: 44-191-219-4747; e-mail: miodrag.stojkovic@ncl.ac.uk. _! o8 n; l" C3 F
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ABSTRACT0 g4 e4 N" U& X! Y! ^( Z
3 n) H5 |" s. E5 }Human embryonic stem cells (hESCs) have been derived from the inner cell mass (ICM) of day-5 through -8 blastocysts . To date, derivation and propagation of undifferentiated hESCs requires plating of both ICM and hESC colonies on mouse embryonic fibroblast (MEF) cells or human feeder (HFF) cells , and this limits the large-scale culture and genetic manipulation of hESCs . To overcome these obstacles, feeder-free systems have been introduced in which hESCs can be grown on different matrices with addition of MEF-conditioned medium or hESC medium supplemented with serum replacement, different growth factors, or in the presence of 6-bromoindirubin-3'-oxime, a specific pharmacological inhibitor of glycogen synthase kinase-3 ./ w1 o! C- K' S8 X
6 A8 S, V$ R8 J0 ^! d M% _One of the most frequently used matrices for feeder-free growth of undifferentiated hESCs is Matrigel, which supports attachment and growth of undifferentiated hESCs in the presence of MEF-conditioned medium . Matrigel is ananimal basement membrane preparation extracted from Engelbreth-Holm-Swarm mouse sarcoma, a tumor rich in extracellular matrix (ECM) proteins: laminin, collagen IV, heparan sulfate proteoglycans, entactin, and nidogen 1. Unfortunately, application of Matrigel or MEF-conditioned medium is not ideal for potential medical application of hESCs because xenogeneic pathogens can be transmitted through culture conditions .. x: N; d$ M9 Z7 g [4 N9 t; h
Q' N3 W+ N6 t! Q+ vWe previously demonstrated that hESCs could be successfully grown on Matrigel with addition of medium conditioned by the fibroblasts derived from differentiated hESCs (hES-dF). In this manuscript, we evaluated whether human serum (HS) could be successfully used as a matrix to help the attachment and growth of hESCs with the aim to create feeder-free and more patient-friendly conditions for the long-term growth of undifferentiated hESCs. We demonstrate here that HS and medium conditioned by hES-dF reduce exposure of hESCs to animal ingredients and provide a safer direction toward completely animal-free conditions for application, handling, and understanding of hESC biology. At the same time, hESCs grown under these conditions maintain all hESC features after prolonged culture, including the developmental potential to differentiate into representative tissues of all three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype.( L. j2 d2 L; T( ~% c" F* @
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MATERIALS AND METHODS , k1 }8 V6 H! _ x. X& ^! v* @: k. q4 M/ A; [% L. c x) f% l, N
Surfaces of culture plates coated with Matrigel or HS were investigated using scanning electron microscopy. This analysis revealed that coating with Matrigel or HS provides a substratum with similar shape (Figs. 1A, 1B), which assists cell adhesion and supports undifferentiated growth of both hESC lines, hES-NCL1 and H1 (Figs. 1C, 1D). When transferred on uncoated plates, hESC colonies do not attach; however, they do form embryoid bodies (not shown) or attach and spontaneously differentiate. On the contrary, both lines cultured on HS and in the presence of hES-dF medium kept their pluripotency for over 27 passages. We found that undifferentiated hESCs grown on HS show typical morphology, that is, small cells with prominent nucleoli (Figs. 2A, 2B) expressing typical cell-surface and intracellular hESC markers: TRA-1-60 (Fig. 2C), TRA-1-81 (Fig. 2D), SSEA-4 (Fig. 2E), AP (Fig. 2F), and OCT-4 (Fig. 2G). When hESCs of both cell lines were grown on HS and in the absence of hES-dF medium, spontaneous differentiation was observed 48 hours after passaging (Fig. 2I). RT-PCR analysis of undifferentiated hESCs showed positive expression of OCT-4, REX-1, NANOG, and TERT (Fig. 2J). Comparative flow cytometry analysis revealed that hES-NCL1 cells grown on Matrigel or HS for 21 passages expressed 79.1% and 81.5% of TRA-1-81 antigen, respectively (data not shown).6 q7 R: | s2 Y3 Y1 a
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Figure 1. Environmental SEM of dishes coated with (A) Matrigel or (B) HS and SEM of hES-NCL1 cells grown on (C) HS or on (D) Matrigel. Note the crystal-like structure in both coated plates and similar morphology, that is, flat surface of the hESCs grown on HS (C; 4-day-old colony, passage 15) or Matrigel (D; 6-day-old colony, passage 14) in the presence of hES-dF–conditioned medium. Spontaneously differentiated hESCs and surface of noncoated plastic dish are presented in (E) and (F), respectively. (E): Note the diversity in the shape of different spontaneously differentiated hESCs. Bars: 20 μm (A, B, E); 50 μm (F); 200 μm (C, D). Abbreviations: hESC, human embryonic stem cell; hES-dF, differentiated hESC; HS, human serum; SEM, scanning electron microscopy.8 ?, k; U4 S b" W5 K6 @3 j' x% a2 w
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Figure 2. Morphology and characterization of (A–E; passage 13) hES-NCL1 and (D–H; passage 15) H1 cells grown on HS in the presence of hES-dF–conditioned medium. (B): Higher magnification of the hESC colony. Note typical morphology of hESCs, that is, high nucleus to cytoplasm ratio, presence of nucleoli. hESCs grown on HS and hES-dF–conditioned medium stained with antibody recognizing the (C) TRA-1-60, (D) TRA-1-81, (E) SSEA-4, (F) alkaline phosphates, and (G) OCT-4 epitopes. (H): Negative OCT-4 control. (I): H1 cells grown on HS in the absence of hES-dF but in the presence of ES medium spontaneously differentiated already after 2 days. (J): RT-PCR analysis of undifferentiated hES-NCL1 (passage 16) cells grown on HS. RT-PCR was proceeded with ( ) or without (–) reverse transcription. PCR products obtained using primers specific for OCT-4, REX1, TERT, NANOG, and GAPDH. (C, D): Red color represents cell nuclei stained with propidium iodide. Bars: 25 μm (B); 50 μm (C); 100 μm (A, D, E); 200 μm (F, G, H, I). Abbreviations: hESC, human embryonic stem cell; hES-dF, differentiated hESC; HS, human serum; RT-PCR, reverse transcription–polymerase chain reaction.( S/ t! b! y3 N/ @
- G0 n% f6 K1 \0 ]Karyotyping of the hESCs showed that both lines hES-NCL1 and H1 grown on HS in the presence of hES-dF–conditioned medium have a normal female or male karyotype (Figs. 3A, 3B), keeping their genomic stability even after 21 passages.! {2 [" y- }2 G, Q. k4 U
0 H s9 Y/ n) D: r3 nFigure 3. Karyotyped (A) hES-NCL1 and (B) H1 (both passage 17) cells grown on HS in the presence of hES-dF–conditioned medium keep their stability and show a normal female and male karyotype, respectively. Abbreviations: hES-dF, differentiated hESC; HS, human serum. 1 x# ^2 r; k* h1 Y- F' V9 D3 w( t/ O8 e
When hES-NCL1 or H1 cells grown on HS in the presence of hES-dF–conditioned medium were not replated after 4–6 days (see Material and Methods), spontaneous differentiation into neuronal precursor, fat cells, cardiomyocytes, and endoderm-like cells was observed (Figs. 4A–4D). The presence of the cells of all three germ lineages was confirmed by RT-PCR, in which expression of cytokeratin 3 (CK3), cytokeratin 19 (CK19) PAX6, NESTIN, GATA4, and Indian hedgehog genes was observed (Fig. 4E). This demonstrates that both hESC lines cultured on HS have the potential to spontaneously differentiate into cells of all three germ layers under in vitro conditions. PCR analysis of undifferentiated hESCs using the same markers as for differentiated hESCs showed only expression of CK19 (Fig. 4F)." D" u$ ^' S o" B
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Figure 4. Spontaneous differentiation of hES-NCL1 and H1 cells grown on HS in the presence of hES-dF–conditioned medium. hES-NCL1 (passage 19) spontaneously differentiates into (A) neuronal, (B) fat, (C) contracting cardiac muscle, and (D) endoderm-like cells, demonstrating their differentiation ability under our in vitro growth conditions. Green color represents cells stained with (A) tubulin ? III, (C) -actin sarcomeric, and (D) -fetoprotein antibodies. (B): Red color represents fat cells stained with oil red O staining.(A, C, D):Blue color represents nuclei stained with Hoechst. (E): RT-PCR analysis of differentiated H1 (passage 16) cells grown on HS demonstrating differentiation ability of hESCs into all three germ layers. PCR products obtained using primers specific for cytokeratin 3 (CK3), cytokeratin 19(CK19), Pax6, Nestin, Gata4, Indian hed gehog (IHH), and GAPDH genes. (F): Undifferentiated H1 cell expressed only CK19. Scale bars: 100 μm (A); 50 μm (B, D); 25 μm (C). Abbreviations: hESC, human embryonic stem cell; hES-dF, differentiated hESC; HS, human serum; RT-PCR, reverse transcription–polymerase chain reaction. . C. a9 q* }" t; f/ w2 u O8 q" S% [% e" a, ]
Under in vivo conditions, hES-NCL1 grown on HS and in the presence of hES-dF–conditioned medium consistently developed into teratomas when grafted into SCID mice. The teratomas were always restricted to the site of transplantation. Gross analysis of excised tumor tissues showed solid teratomas and lesions containing fluid-filled cystic masses accompanied with solid tissues. Histological examination of teratomas revealed advanced differentiation of structures representative of all three embryonic germ layers, including cartilage, muscle, primitive neuroectoderm, neural ganglia, kidney, secretory epithelia, and connective tissues (Figs. 5A–5F). Moreover, such tissues formed complex arrangements, recapitulating the development of complex structures that no doubt require coordinated interactions between different cell types derived from different germ layers.% E+ N. w+ |& P
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Figure 5. Histological analysis of teratomas formed from grafted colonies of human feeder–independent hES-NCL1 cells in SCID mice. (A): General structure of teratoma showing a range of different tissue types, including cartilage (cart), muscle (mus) and epithelia (ep). (B): Higher magnification example of cartilage (cart) and an adjacent neural element (ne). (C): Wall of intestinal tract showing epithelium (ep), mucosa (m), smooth muscle layer (mus), submucosal glands (sg), and neural elements (ne). (D): Higher magnification image of intestinal mucosa (m). The epithelium is typical of that found in the large intestine and consists of a single layer of columnar cells that primarily secrete mucous. (E): Longitudinal section through smooth muscle (mus). (F): Kidney tissue including glomerulus (glom) with surrounding Bowman’s space and adjacent tubules (tub). Note the vascular pole of the glomerulus and the presence of a blood vessel (bv). Histological staining: (A–C, E) Weigert’s and (D, F) hematoxylin and eosin. Scale bars: 500 μm (A); 100 μm (B, D–F); 200 μm (C). Abbreviation: SCID, severe combined immunodeficient.7 d5 S) K1 M; t6 b
7 O" u% g0 i+ Y+ _3 W: F! TDISCUSSION % s% t, L% n5 b# F& G' ] 3 S$ R5 Z, n% K2 wThe authors would like to thank Vivian Thompson, Tracey Scott-Davey (Biomedical EM Unit, Newcastle University, Newcastle upon Tyne), and Grant Staines (SAgE Faculty Services, Newcastle University) for SEM and ESEM analysis. This work was supported by Newcastle University Hospitals Special Trustees, One NorthEast Regional Development Agency (Newcastle), Newcastle Health Charity, the Department of Health, and Medical Research Council, London, grant No. G0301182.4 D) I6 K) r' [& L
3 a8 Q A& y. J4 eREFERENCES. I. s) q; l, N7 I3 p/ `1 z/ u* P
. h! r$ `( O( `+ ?# uThomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell line from human blastocysts. Science 1998;282:1145–1147., Q3 a/ v1 m. b: P/ y
. h% y* o7 ?$ a M" m
Reubinoff BE, Pera MF, Fong C-Y et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000;18:399–404.' l, h9 r) Z) @) [- R4 e8 n( S
8 S( y% G/ D F7 bRichards M, Fong C-Y, Chan W-K et al. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cell lines. Nat Biotechnol 2002;20:933–936. / m1 |: a- u; N0 r& r1 w' ~" K- \1 f2 m5 l
Hovatta O, Mikkola M, Gertow K et al. A culture system using foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod 2003;18:1404–1409. % m5 V, |4 h% y* z( K( X. `0 }" ]5 z0 d- M! F4 E4 g
Mitalipova M, Calhoun J, Shin S et al. Human embryonic stem cell lines derived from discarded embryos. STEM CELLS 2003;21:521–526. : U* m D: i( B$ R" { ) ?/ X0 Z% _3 F, n7 D+ QPickering SJ, Braude PR, Patel M et al. Preimplantation genetic diagnosis as a novel source of embryos for stem cell research. RBM Online 2003;7:353–364.% P& i7 p1 W5 H4 A, s6 _
+ b3 V0 g+ `$ e" j; Q" ^, TCowan CA, Klimanskaya I, McMahon J et al. Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med 2004;350:1353–1356.6 F3 c' v# r- s5 s5 ^
) R* G( E: a: fPark JH, 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. ) p7 t8 S! z9 O) ~# s# R/ l , s( Y; X1 ]$ a# ^- b9 l* jStojkovic M, Lako M, Stojkovic P et al. Derivation of human embryonic stem cells from day 8 blastocysts recovered after three-step in vitro culture. STEM CELLS 2004;22:790–797.9 Y4 g+ Y* o! P5 [; U9 @4 \
$ Q; D# ]8 K4 d. |2 LAmit M, Margulets V, Segev H et al. Human feeder layers for human embryonic stem cells. Biol Reprod 2003;68:2150–2156.( }; V; N$ N/ U+ j) S2 z
. C6 T9 F4 A6 n" y! h9 l% K* U$ g# ~' ^
Cheng L, Hammond H, Zhaohui Y et al. Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture. STEM CELLS 2003;21:131–142., P4 J1 L3 B6 ?6 k' f2 Z
* P- u0 i8 b9 M* [' L TRichards M, Tan S, Fong C-Y et al. Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells. STEM CELLS 2003;21:546–556.: F; X& Y, U6 w6 \: H% ^' @5 ~. W
9 y' L7 \. s, I3 `8 WZwaka TP, Thomson JA. Homologous recombination in human embryonic stem cells. Nat Biotechnol 2003;21:319–321. # E$ ~. y8 |2 i & g* y4 h7 P- I9 ]Xu C, Inokuma MS, Denham J et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001;19:971–974. & U3 f* j$ W6 h& o. w6 O" \, X) Y! P6 Q
Rosler ES, Fisk GJ, Ares X et al. Long-term culture of human embryonic stem cells in feeder-free conditions. Dev Dyn 2004;229:259–274. 2 X3 M4 [2 p) }4 p8 \3 [$ _ , c7 u2 q0 F8 a$ x' V, v# mCarpenter MK, Rosler ES, Fisk GJ et al. Properties of four human embryonic stem cell lines maintained in a feeder-free culture system. Dev Dyn 2004;229:243–258.. x" @: T! U9 \7 {2 _1 {/ D7 L
' M! k4 _7 z% P6 Y( ^- c( G5 H
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./ B8 w7 w- j; _, u1 @: R
0 J- I, s, O6 n% nSatoN, Meijer L, Skaltsounis L et al. 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.. X$ a4 u! X. _( `4 h% G
! g0 D- H' n1 A) H- }$ o: o8 \
Draper JS, Moore HD, Ruban LN et al. Culture and characterization of human embryonic stem cells. Stem Cells Dev 2004;13:325–336.: h( U) B9 l1 j# O" u% [
- U1 I# M- i$ ?, Q( p' Q
Xu R-H, Peck RM, Li D et al. Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nat Methods 2005;2:185–190. + O- t( P1 V* N - m2 V' u+ _! L# C) v& ~9 E: S# y5 XXu C, Rosler E, Jiang J et al. Basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth without conditioned medium. STEM CELLS 2005;23:315–323.# L$ ?$ |+ w9 b* x) z# d
1 N9 u) a1 k ?Klimanskaya I, Chung Y, Meisner L et al. Human embryonic stem cells derived without feeder cells. Available at http://www.thelancet.com/journals/lancet/full?volume=365&issue=9471. Accessed May 20, 2005.2 q7 i1 {8 l+ N
5 H0 }% } O7 I. u; m) _Stojkovic P, Lako M, Stewart R et al. An autogeneic feeder-system that efficiently supports undifferentiated growth of human embryonic stem cells. STEM CELLS 2005;23:306–314. 3 m# K3 K- @6 \9 \2 X1 j4 I$ s% [' ?5 U" k+ [( D- ?
Uhm JH, Dooley NP, Kyritsis AP et al. Vitronectin, a glioma-derived extracellular matrix protein, protects tumor cells from apoptotic death. Clin Cancer Res 1999;5:1587–1594.. Y& Y, ^0 n" V0 r+ U5 c
+ ~* w. i0 ]$ W- g! J# iLim JW, Bodnar A. Proteome analysis of conditioned medium from mouse embryonic fibroblast feeder layers which support the growth of human embryonic stem cells. Proteomics 2002;2:1187–1203.2 t& h+ e; I* X- P" n, k/ E- R
* W+ h; c" d- C" z& R7 V6 ]Choo AB, Padmanabhan J, Chin AC et al. Expansion of pluripotent human embryonic stem cells on human feeders. Biotechnol Bioeng 2004;88:321–331. + o6 {. i7 i7 A: R2 N5 Y7 P2 I6 a
Ikari Y, Mulvihill E, Schwartz SM. Alpha 1-Proteinase Inhibitor, alpha 1-antichymotrypsin, and alpha 2-macroglobulin are the antiapoptotic factors of vascular smooth muscle cells. J Cell Biol 2000;149:741–754.3 k; A. m, ]& p+ H3 r
2 I+ J* C5 y- b5 N9 j/ x& D0 YAlmeida EC, Ilic D, Han Q et al. Effects of growth factors and extracellular matrix on survival of human airway smooth muscle cells. Am J Respir Cell Mol Biol 2001;25:569–576.( W( j8 E. H& `6 V$ o7 o. l1 D5 g
. o* O! N+ \ o5 V4 g; G
Li X, Talts U, Talts JF et al. Akt/PKB regulates laminin and collagen IV isotypes of the basement membrane. Proc Natl Acad Sci U S A 2001;98:14416–14421. : p/ f: L; J, N4 B; d6 r0 I) E& b; ~ g2 p, I9 {' Y
Schuldiner M, Yanuka O, Itskovitz-Eldor J et al. Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc Natl Acad Sci U S A 2000;97:11307–11312.(Petra Stojkovica, Majlind)作者: wangabu 时间: 2009-4-19 08:18